Checkpoint Inhibitor and Vaccine Combinations and Use of Same For Immunotherapy

ABSTRACT

Disclosed herein is a vaccine comprising an antigen and checkpoint inhibitor. Also disclosed herein is a method for enhancing an immune response in a subject. The method may comprise administering the vaccine to the subject in need thereof.

TECHNICAL FIELD

The present invention relates to vaccines combined with checkpointinhibitor antibodies, and use of such combination for immunotherapy.

BACKGROUND

Vaccines are used to stimulate an immune response in an individual toprovide protection against and/or treatment for a particular disease.Some vaccines include an antigen to induce the immune response. Someantigens elicit a strong immune response while other antigens elicit aweak immune response. A weak immune response to an antigen can bestrengthened by including an adjuvant in the vaccine. Adjuvants come inmany different forms, for example, aluminum salts, oil emulsions,sterile constituents of bacteria or other pathogens, cytokines, and soforth.

Programmed cell death protein 1 also known as PD-1 is a 288 amino acidcell surface protein molecule that in humans is encoded by the PDCD1gene. This protein is expressed in pro-B cells and is thought to play arole in their differentiation. PD1 is a type I membrane protein of 268amino acids and a member of the extended CD28/CTLA-4 family of T cellregulators. The protein's structure includes an extracellular IgV domainfollowed by a transmembrane region and an intracellular tail. Theintracellular tail contains two phosphorylation sites located in animmunoreceptor tyrosine-based inhibitory motif and an immunoreceptortyrosine-based switch motif, which suggests that PD-1 negativelyregulates TCR signals. PD-1 has two ligands, PD-L1 and PD-L2, which aremembers of the B7 family. PD-L1 protein is upregulated on macrophagesand dendritic cells (DC) in response to LPS and GM-CSF treatment, and onT cells and B cells upon TCR and B cell receptor signaling, whereas inresting mice, PD-L1 mRNA can be detected in the heart, lung, thymus,spleen, and kidney. PD-L1 is expressed on almost all murine tumor celllines, including PA1 myeloma, P815 mastocytoma, and B16 melanoma upontreatment with IFN-γ. PD-L2 expression is more restricted and isexpressed mainly by DCs and a few tumor lines.

There are studies suggesting that PD-1 and its ligands negativelyregulate immune responses. PD-1 knockout mice have been shown to developlupus-like glomerulonephritis and dilated cardiomyopathy on the C57BL/6and BALB/c backgrounds, respectively. In vitro, treatment of anti-CD3stimulated T cells with PD-L1-Ig results in reduced T cell proliferationand IFN-γ secretion. It appears that upregulation of PD-L1 may allowcancers to evade the host immune system. PD-L1 expression has been shownto correlate inversely with intraepithelial CD8+ T-lymphocyte count,suggesting that PD-L1 on tumor cells may suppress antitumor CD8+ Tcells.

LAG3 and TIM3 are some of the many receptor molecules on the surface ofT lymphocytes that exert inhibitory functions.

T cell immunoglobulin domain and mucin domain 3 (TIM-3; also known asHAVCR2), is a human protein that is encoded by the HAVCR2 gene. TIM-3 isa protein surface receptor expressed by activated T cells of theIFNgamma-producing CD4 Th1 and CD8 cytotoxic T cells. Its ligand isgalectin-9 which is abundantly expressed in the tumor microenvironmentinduces cell death and T cell exhaustion of CD4 and CD8 T cells.Evidence of Tim-3 as a key immune checkpoint in either tumor orviral-induced immune suppression comes from demonstration that Tim-3expressing CD8 T cells are the most suppressed or dysfunctionalpopulation of CD8 T cell in preclinical models.

Lymphocyte activation gene 3 (Lag-3 also known as CD223) is a member ofthe Ig superfamily that is expressed only on activated and tolerized Tcells that binds MHC-II molecules and which is known to transduceinhibitory signals. LAG-3 is markedly upregulated on exhausted T cellscompared to effector or memory T cells. LAG-3 negatively regulates Tcell expansion by inhibiting T cell receptor induced calcium fluxes,thus controlling the size of the T cell memory pool. Studies have shownthat in the context of cancer, LAG3 is upregulated on TILs and blockadeof LAG-3 can enhance antitumor T cell immune responses. Blockage ofLAG-3 in a viral chronic model that evokes CD8 T cells exhaustion, caninvograte the CD8 T cell responses.

Collectively, these aforementioned proteins, along with other inhibitoryreceptors, such as CTLA-4, are important players in the CD8 T cellexhaustion that takes place in chronic immune conditions such as chronicviral infection and cancer in both experimental models and humans. Theseknown features and function of PD1-1, CTLA-4, TIM-3 and LAG-3 make theman appealing target for immune modulation in vaccine settings.

Vaccines are also administered in many different ways (e.g., injection,orally, etc.) into many different tissues (e.g., intramuscular, nasal,etc.). Not all delivery methods, however, are equal. Some deliverymethods allow for greater compliance within a population of individualswhile other delivery methods may affect immunogenicity and/or safety ofthe vaccine.

Accordingly, there remains a need for more effective immunotherapy usingsynthetic antigens combined with checkpoint inhibitors, in particularTIM-3, and LAG-3. In addition, there remains a need for improvedtreatment methods to improve the immune response generated fromcombination of checkpoint inhibitors and synthetic antigens.

SUMMARY OF THE PREFERRED EMBODIMENTS

Aspects of the present invention include compositions for enhancing animmune response against an antigen in a subject in need thereof,comprising TIM-3antibody or LAG-3 antibody in combination with asynthetic antigen capable of generating an immune response in thesubject, or a biologically functional fragment or variant thereof.

The synthetic antigen can be an isolated DNA that encodes for theantigen

Preferably, the synthetic antigen can be selected from the groupconsisting of: hTERT, prostate, WT1, tyrosinase, NYES01, PRAME, MAGE,CMV, herpes, HIV, HPV, HCV, HBV, influenza, RSV, Plasmodium falciparum,and C. difficle.

The compositions provided herein can also include a pharmaceuticallyacceptable excipient.

Aspects of the invention also include methods for increasing an immuneresponse in a subject in need thereof by administering any of thecompositions provided herein to the subject. The methods of increasingan immune response can also include an electroporating step.

Further aspects of the invention include methods of administering acheckpoint inhibitor in combination with an antigen to enhance theimmune response of the antigen, where the checkpoint inhibitor isdelivered after a prime and boost administering of the antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides graphs that show a 30% increase in T cell responsesinduced by co-therapy with anti PDL1 antibody and HPV vaccines.

FIG. 2. Provides plot graphs that depict the total hTERT-specific CD8+ Tcells expressing total IFNγ for mice treated with PD1. The left plotgraph show the percentages of hTERT-specific CD3+CD8+ T cells displayingdouble release of the cytokines IFNγ and TNFα. Experiments wereperformed independently at least twice and data represent the mean±SEMof four mice per group.

FIG. 3. Provides plot graphs that depict the total hTERT-specific CD8+ Tcells expressing total IFNγ for mice treated with TIM3. The left plotgraph show the percentages of hTERT-specific CD3+CD8+ T cells displayingdouble release of the cytokines IFNγ and TNFα. Experiments wereperformed independently at least twice and data represent the mean±SEMof four mice per group.

FIG. 4. Provides plot graphs that depict the total hTERT-specific CD8+ Tcells expressing total IFNγ for mice treated with LAG3. The left plotgraph show the percentages of hTERT-specific CD3+CD8+ T cells displayingdouble release of the cytokines IFNγ and TNFα. Experiments wereperformed independently at least twice and data represent the mean±SEMof four mice per group.

FIG. 5. Provides flow cytometry results for early delivery of mAbcheckpoints. (A) shows plot graphs that depict the hTERT-specific CD8 Tcells expressing total IFNγ for mice treated with or without PD1, TIM3and LAG3 soon after priming immunization. (B) shows plot graphs thatdepict the hTERT-specific CD8 T cells expressing total IFNγ for micetreated with or without PD1, TIM3 and LAG3 soon after boostimmunization.

FIG. 6. Provides flow cytometry results for late delivery of mAbcheckpoints. (A) shows plot graphs that depict the hTERT-specific CD8 Tcells expressing total IFNγ for mice treated with or without PD1, TIM3and LAG3 soon after priming immunization. (B) shows plot graphs thatdepict the hTERT-specific CD8 T cells expressing total IFNγ for micetreated with or without PD1, TIM3 and LAG3 soon after boostimmunization.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to a vaccine that can be used to increaseor enhance an immune response, ie., create a more effective immuneresponse, by combining a vaccine, in many cases a synthetic antigen,with a checkpoint inhibitor, in particular PD1, PDL1, TIM-3, and LAG-3antibodies. In some instances, the antibodies, in particular TIM-3antibodies and LAG-3 antibodies, can be administered in combination withthe antigen; whereas, in other instances, the antibodies, in particularTIM-3 antibodies and LAG-3 antibodies, can be administered separatelyfrom the antigen of the vaccine. In some instances the antibodies, inparticular TIM-3 antibodies and LAG-3 antibodies, comprise a DNAsequence that encodes such antibody, which includes at least thevariable regions of the immunoglobulin.

The vaccine of the present invention can increase the immune response tothe antigen in the subject by increasing the CD8⁺ T cell response ascompared to the vaccine not including checkpoint inhibitors. Thisincreased CD8⁺ T cell response has cytolytic activity and secretes theanti-viral cytokine interferon-gamma (IFN-γ).

Aspects of the present invention include compositions for enhancing animmune response against an antigen in a subject in need thereof,comprising TIM-3antibody or LAG-3 antibody in combination with asynthetic antigen capable of generating an immune response in thesubject, or a biologically functional fragment or variant thereof.

The synthetic antigen can be an isolated DNA that encodes for theantigen

Preferably, the synthetic antigen can be selected from the groupconsisting of: hTERT, prostate, WT1, tyrosinase, NYES01, PRAME, MAGE,CMV, herpes, HIV, HPV, HCV, HBV, influenza, RSV, Plasmodium falciparum,and C. difficle.

The HPV antigen can be E6 and E7 domains of subtypes selected from thegroup consisting of: HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV52, andHPV58, and a combination thereof.

The HIV antigen can be selected from the group consisting of: Env A, EnvB, Env C, Env D, B Nef-Rev, and Gag, and a combination thereof.

The influenza antigen can be selected from the group consisting of: H1HA, H2 HA, H3 HA, H5 HA, BHA antigen, and any combination thereof.

The Plasmodium falciparum antigen includes a circumsporozoite (CS)antigen.

The C. difficle antigen can be selected from the group consisting of:Toxin A, and Toxin B, and a combination thereof.

The HCV antigen can be selected from the group consisting of: E1, E2,NS3, NS4a, NS4b, NS5a, and NS5b, and a combination thereof.

The HBV antigen can be selected from the group consisting of: surfaceantigen type A, surface antigen type B, surface antigen type C, surfaceantigen type D, surface antigen type E, surface antigen type F, surfaceantigen type G, surface antigen type H, and core antigen, and acombination thereof.

The RSV antigen can be selected from the group consisting of: F, G, NS1,NS2, N, M, M2-1, M2-2, P, SH, and L protein, and a combination thereof.

The synthetic antigen can be hTERT, WT1 antigen, tyrosinase, NYES01, orPRAME.

The prostate antigen can be selected from the group consisting of: PSA,PSMA, STEAP, PSCA, and PAP, and a combination thereof.

The compositions provided herein can also include a pharmaceuticallyacceptable excipient.

Aspects of the invention also include methods for increasing an immuneresponse in a subject in need thereof by administering any of thecompositions provided herein to the subject. The methods of increasingan immune response can also include an electroporating step.

1. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

“Adjuvant” as used herein means any molecule added to the vaccinedescribed herein to enhance the immunogenicity of the antigens, and inparticular refers to checkpoint inhibitor antibodies.

“Checkpoint inhibitor” as used herein means are inhibitors or moleculesthat block immune checkpoints as commonly understood in the field ofcancer immunotherapy. More commonly the checkpoint inhibitors areantibodies that block these immune checkpoints, such as PD1 (on T cell)to its ligand PDL1 (on dendritic cell). Some examples of knowncheckpoint inhibitors include ipilimumab, pembrolizumab, nivolumab,pidilizumab, and others.

“Coding sequence” or “encoding nucleic acid” as used herein means thenucleic acids (RNA or DNA molecule) that comprise a nucleotide sequencewhich encodes a protein. The coding sequence can further includeinitiation and termination signals operably linked to regulatoryelements including a promoter and polyadenylation signal capable ofdirecting expression in the cells of an individual or mammal to whichthe nucleic acid is administered.

“Complement” or “complementary” as used herein means a nucleic acid canmean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairingbetween nucleotides or nucleotide analogs of nucleic acid molecules.

“Electroporation,” “electro-permeabilization,” or “electro-kineticenhancement” (“EP”) as used interchangeably herein means the use of atransmembrane electric field pulse to induce microscopic pathways(pores) in a bio-membrane; their presence allows biomolecules such asplasmids, oligonucleotides, siRNA, drugs, ions, and water to pass fromone side of the cellular membrane to the other.

“Fragment” or “immunogenic fragment” as used herein means a nucleic acidsequence or a portion thereof that encodes a polypeptide capable ofeliciting an immune response in a mammal. The fragments can be DNAfragments selected from at least one of the various nucleotide sequencesthat encode protein fragments set forth below. Fragments can comprise atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95% ofone or more of the nucleic acid sequences set forth below. In someembodiments, fragments can comprise at least 20 nucleotides or more, atleast 30 nucleotides or more, at least 40 nucleotides or more, at least50 nucleotides or more, at least 60 nucleotides or more, at least 70nucleotides or more, at least 80 nucleotides or more, at least 90nucleotides or more, at least 100 nucleotides or more, at least 150nucleotides or more, at least 200 nucleotides or more, at least 250nucleotides or more, at least 300 nucleotides or more, at least 350nucleotides or more, at least 400 nucleotides or more, at least 450nucleotides or more, at least 500 nucleotides or more, at least 550nucleotides or more, at least 600 nucleotides or more, at least 650nucleotides or more, at least 700 nucleotides or more, at least 750nucleotides or more, at least 800 nucleotides or more, at least 850nucleotides or more, at least 900 nucleotides or more, at least 950nucleotides or more, or at least 1000 nucleotides or more of at leastone of the nucleic acid sequences set forth below.

Fragment or immunogenic fragment as used herein also means a polypeptidesequence or a portion thereof that is capable of eliciting an immuneresponse in a mammal. The fragments can be polypeptide fragmentsselected from at least one of the various amino acid sequence set forthbelow. Fragments can comprise at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 95% of one or more of the proteins set forthbelow. In some embodiments, fragments can comprise at least 20 aminoacids or more, at least 30 amino acids or more, at least 40 amino acidsor more, at least 50 amino acids or more, at least 60 amino acids ormore, at least 70 amino acids or more, at least 80 amino acids or more,at least 90 amino acids or more, at least 100 amino acids or more, atleast 110 amino acids or more, at least 120 amino acids or more, atleast 130 amino acids or more, at least 140 amino acids or more, atleast 150 amino acids or more, at least 160 amino acids or more, atleast 170 amino acids or more, at least 180 amino acids or more, atleast 190 amino acids or more, at least 200 amino acids or more, atleast 210 amino acids or more, at least 220 amino acids or more, atleast 230 amino acids or more, or at least 240 amino acids or more of atleast one of the proteins set forth below.

“Genetic construct” as used herein refers to the DNA or RNA moleculesthat comprise a nucleotide sequence which encodes a protein. The codingsequence includes initiation and termination signals operably linked toregulatory elements including a promoter and polyadenylation signalcapable of directing expression in the cells of the individual to whomthe nucleic acid molecule is administered. As used herein, the term“expressible form” refers to gene constructs that contain the necessaryregulatory elements operable linked to a coding sequence that encodes aprotein such that when present in the cell of the individual, the codingsequence will be expressed.

“Identical” or “identity” as used herein in the context of two or morenucleic acids or polypeptide sequences, means that the sequences have aspecified percentage of residues that are the same over a specifiedregion. The percentage can be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) can be considered equivalent.Identity can be performed manually or by using a computer sequencealgorithm such as BLAST or BLAST 2.0.

“Immune response” as used herein means the activation of a host's immunesystem, e.g., that of a mammal, in response to the introduction ofantigen. The immune response can be in the form of a cellular or humoralresponse, or both.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” as used hereinmeans at least two nucleotides covalently linked together. The depictionof a single strand also defines the sequence of the complementarystrand. Thus, a nucleic acid also encompasses the complementary strandof a depicted single strand. Many variants of a nucleic acid can be usedfor the same purpose as a given nucleic acid. Thus, a nucleic acid alsoencompasses substantially identical nucleic acids and complementsthereof. A single strand provides a probe that can hybridize to a targetsequence under stringent hybridization conditions. Thus, a nucleic acidalso encompasses a probe that hybridizes under stringent hybridizationconditions.

Nucleic acids can be single stranded or double stranded, or can containportions of both double stranded and single stranded sequence. Thenucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid can contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids can be obtained by chemical synthesismethods or by recombinant methods.

“Operably linked” as used herein means that expression of a gene isunder the control of a promoter with which it is spatially connected. Apromoter can be positioned 5′ (upstream) or 3′ (downstream) of a geneunder its control. The distance between the promoter and a gene can beapproximately the same as the distance between that promoter and thegene it controls in the gene from which the promoter is derived. As isknown in the art, variation in this distance can be accommodated withoutloss of promoter function.

A “peptide,” “protein,” or “polypeptide” as used herein can mean alinked sequence of amino acids and can be natural, synthetic, or amodification or combination of natural and synthetic.

“Promoter” as used herein means a synthetic or naturally-derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter can comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of same. A promoter can also comprise distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A promoter can bederived from sources including viral, bacterial, fungal, plants,insects, and animals. A promoter can regulate the expression of a genecomponent constitutively or differentially with respect to cell, thetissue or organ in which expression occurs or, with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents. Representative examples of promoters include thebacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lacoperator-promoter, tac promoter, SV40 late promoter, SV40 earlypromoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40late promoter and the CMV IE promoter.

“Signal peptide” and “leader sequence” are used interchangeably hereinand refer to an amino acid sequence that can be linked at the aminoterminus of a synthetic antigen, including some of the examples citedherein. Signal peptides/leader sequences typically direct localizationof a protein. Signal peptides/leader sequences used herein preferablyfacilitate secretion of the protein from the cell in which it isproduced. Signal peptides/leader sequences are often cleaved from theremainder of the protein, often referred to as the mature protein, uponsecretion from the cell. Signal peptides/leader sequences are linked atthe N terminus of the protein.

“Subject” as used herein can mean a mammal that wants to or is in needof being immunized with the herein described vaccine. The mammal can bea human, chimpanzee, dog, cat, horse, cow, pig, chicken mouse, or rat.

“Substantially identical” as used herein can mean that a first andsecond amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%,or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 1100 or more amino acids. Substantially identicalcan also mean that a first nucleic acid sequence and a second nucleicacid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1100 or more nucleotides.

“Treatment” or “treating,” as used herein can mean protecting of ananimal from a disease through means of preventing, suppressing,repressing, or completely eliminating the disease. Preventing thedisease involves administering a vaccine of the present invention to ananimal prior to onset of the disease. Suppressing the disease involvesadministering a vaccine of the present invention to an animal afterinduction of the disease but before its clinical appearance. Repressingthe disease involves administering a vaccine of the present invention toan animal after clinical appearance of the disease.

“Variant” used herein with respect to a nucleic acid means (i) a portionor fragment of a referenced nucleotide sequence; (ii) the complement ofa referenced nucleotide sequence or portion thereof; (iii) a nucleicacid that is substantially identical to a referenced nucleic acid or thecomplement thereof; or (iv) a nucleic acid that hybridizes understringent conditions to the referenced nucleic acid, complement thereof,or a sequences substantially identical thereto.

Variant can further be defined as a peptide or polypeptide that differsin amino acid sequence by the insertion, deletion, or conservativesubstitution of amino acids, but retain at least one biologicalactivity. Representative examples of “biological activity” include theability to be bound by a specific antibody or to promote an immuneresponse. Variant can also mean a protein with an amino acid sequencethat is substantially identical to a referenced protein with an aminoacid sequence that retains at least one biological activity. Aconservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity, degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157:105-132 (1982). The hydropathic index of an amino acid is based on aconsideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indexes of ±2 are substituted. The hydrophilicity of aminoacids can also be used to reveal substitutions that would result inproteins retaining biological function. A consideration of thehydrophilicity of amino acids in the context of a peptide permitscalculation of the greatest local average hydrophilicity of thatpeptide, a useful measure that has been reported to correlate well withantigenicity and immunogenicity. Substitution of amino acids havingsimilar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. Substitutions can be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

A variant may be a nucleic acid sequence that is substantially identicalover the full length of the full gene sequence or a fragment thereof.The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical over the full length of the gene sequence or a fragmentthereof. A variant may be an amino acid sequence that is substantiallyidentical over the full length of the amino acid sequence or fragmentthereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical over the full length of the amino acid sequence or afragment thereof.

“Vector” as used herein means a nucleic acid sequence containing anorigin of replication. A vector can be a viral vector, bacteriophage,bacterial artificial chromosome or yeast artificial chromosome. A vectorcan be a DNA or RNA vector. A vector can be a self-replicatingextrachromosomal vector, and preferably, is a DNA plasmid.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

2. Vaccine

Provided herein are vaccines comprising an antigen and checkpointinhibitors, preferably checkpoint inhibitor antibodies. The antibodiespreferably are PD1 antibody, PDL1 antibody, TIM-3antibody and/or LAG-3antibody. The combination can be a single formulation or can be separateand administered in sequence (either antigen first and then checkpointinhibitor, or checkpoint inhibitor first and then antigen). The vaccinecan increase antigen presentation and the overall immune response to theantigen in a subject. The combination of antigen and checkpointinhibitor induces the immune system more efficiently than a vaccinecomprising the antigen alone. This more efficient immune responseprovides increased efficacy in the treatment and/or prevention of anydisease, in particular cancer, pathogen, or virus.

The antigen and checkpoint inhibitors, preferably are PD1 antibody, PDL1antibody, TIM-3antibody and/or LAG-3 antibody, of the vaccine can beadministered together or separately to the subject in need thereof. Insome instances, the checkpoint inhibitors can be administered separatelyfrom the antigen of the vaccine.

In some embodiments, the checkpoint inhibitors can be administered atleast 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84hours, or 96 hours before or after administration of the antigen to thesubject. In other embodiments, the PD1 antibody or PDL1 antibody can beadministered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60days, or 90 days before or after administration of the antigen to thesubject.

In still other embodiments, the checkpoint inhibitors can beadministered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13weeks, 14 weeks, or 15 weeks before or after administration of theantigen to the subject. In other embodiments, the PD1 antibody or PDL1antibody can be administered about 12 hours to about 15 weeks, about 12hours to about 10 weeks, about 12 hours to about 5 weeks, about 12 hoursto about 1 week, about 12 hours to about 60 hours, about 12 hours toabout 48 hours, about 24 hours to about 15 weeks, about 60 hours toabout 15 weeks, about 96 hours to about 15 weeks, about 1 day to about15 weeks, about 5 days to about 15 weeks, about 10 days to about 15weeks, about 15 days to about 15 weeks, about 20 days to about 15 weeks,about 25 days to about 15 weeks, about 30 days to about 15 weeks, about1 week to about 15 weeks, about 5 weeks to about 15 weeks, or about 10weeks to about 15 weeks before or after administration of the antigen tothe subject.

The vaccine of the present invention can have features required ofeffective vaccines such as being safe so the vaccine itself does notcause illness or death; being protective against illness resulting fromexposure to live pathogens such as viruses or bacteria; inducingneutralizing antibody to prevent infection of cells; inducing protectiveT cell against intracellular pathogens; and providing ease ofadministration, few side effects, biological stability, and low cost perdose. The vaccine can accomplish some or all of these features bycombining the antigen with the checkpoint inhibitors, preferably the PD1antibody, PDL1 antibody, TIM-3antibody and/or LAG-3 antibody asdiscussed below.

The vaccine can further modify epitope presentation within the antigento induce greater immune response to the antigen that a vaccinecomprising the antigen alone. The vaccine can further induce an immuneresponse when administered to different tissues such as the muscle orthe skin.

a. Checkpoint Inhibitors

Checkpoint inhibitors can be any antagonist to the various immunecheckpoints, and are preferably antibodies that block immunecheckpoints. The antibodies can be a protein including a Fab, monoclonalor polyclonal. The antibodies can also be a DNA expression constructthat encodes for and can express functional antibodies. The vaccine canfurther comprise a TIM-3antibody or LAG-3 antibody. The antibody can bea synthetic antibody comprised of DNA sequence encoding at least thevariable regions of an immunoglobulin. Such antibody can be generated byidentifying or screening for the antibody described above, which isreactive to or binds the antigen described above. The method ofidentifying or screening for the antibody can use the antigen inmethodologies known in those skilled in art to identify or screen forthe antibody. Such methodologies can include, but are not limited to,selection of the antibody from a library (e.g., phage display) andimmunization of an animal followed by isolation and/or purification ofthe antibody. See for example methods available in Rajan, S., and Sidhu,S., Methods in Enzymology, vol 502, Chapter One “Simplified SyntheticAntibody Libraries (2012), which is incorporated herein in its entirety.TIM-3and LAG-3 antibodies can also be combined with other checkpointinhibitor antibodies, also including PD1, PDL1, CTLA4, CD40, and others.The checkpoint inhibitors can be a known product such as, for example,nivolumab, pembrolizumab, pidilizumab, BMS-936559 (SeeClinicalTrials.gov Identifier NCT02028403), MPDL3280A (Roche, seeClinicalTrials.gov Identifier NCT02008227), MDX1105-01 (Bristol MyersSquibb, see ClinicalTrials.gov Identifier NCT00729664), MEDI4736(MedImmune, See ClinicalTrials.gov Identifier NCT01693562), and MK-3475(Merck, see ClinicalTrials.gov Identifier NCT02129556).

Synthetic Antibody (DNA Form)

The antibody can be encoded by a nucleic acid sequence (cDNA) thatencodes for the elements as follows:

The antibody can include a heavy chain polypeptide and a light chainpolypeptide. The heavy chain polypeptide can include a variable heavychain (VH) region and/or at least one constant heavy chain (CH) region.The at least one constant heavy chain region can include a constantheavy chain region 1 (CH1), a constant heavy chain region 2 (CH2), and aconstant heavy chain region 3 (CH3), and/or a hinge region.

In some embodiments, the heavy chain polypeptide can include a VH regionand a CH1 region. In other embodiments, the heavy chain polypeptide caninclude a VH region, a CH1 region, a hinge region, a CH2 region, and aCH3 region.

The heavy chain polypeptide can include a complementarity determiningregion (“CDR”) set. The CDR set can contain three hypervariable regionsof the VH region. Proceeding from N-terminus of the heavy chainpolypeptide, these CDRs are denoted “CDR1,” “CDR2,” and “CDR3,”respectively. CDR1, CDR2, and CDR3 of the heavy chain polypeptide cancontribute to binding or recognition of the antigen.

The light chain polypeptide can include a variable light chain (VL)region and/or a constant light chain (CL) region. The light chainpolypeptide can include a complementarity determining region (“CDR”)set. The CDR set can contain three hypervariable regions of the VLregion. Proceeding from N-terminus of the light chain polypeptide, theseCDRs are denoted “CDR1,” “CDR2,” and “CDR3,” respectively. CDR1, CDR2,and CDR3 of the light chain polypeptide can contribute to binding orrecognition of the antigen.

The antibody may comprise a heavy chain and a light chaincomplementarity determining region (“CDR”) set, respectively interposedbetween a heavy chain and a light chain framework (“FR”) set whichprovide support to the CDRs and define the spatial relationship of theCDRs relative to each other. The CDR set may contain three hypervariableregions of a heavy or light chain V region. Proceeding from theN-terminus of a heavy or light chain, these regions are denoted as“CDR1,” “CDR2,” and “CDR3,” respectively. An antigen-binding site,therefore, may include six CDRs, comprising the CDR set from each of aheavy and a light chain V region.

The antibody can be an immunoglobulin (Ig). The Ig can be, for example,IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can include the heavychain polypeptide and the light chain polypeptide. The heavy chainpolypeptide of the immunoglobulin can include a VH region, a CH1 region,a hinge region, a CH2 region, and a CH3 region. The light chainpolypeptide of the immunoglobulin can include a VL region and CL region.

Additionally, the proteolytic enzyme papain preferentially cleaves IgGmolecules to yield several fragments, two of which (the F(ab) fragments)each comprise a covalent heterodimer that includes an intactantigen-binding site. The enzyme pepsin is able to cleave IgG moleculesto provide several fragments, including the F(ab′)₂ fragment, whichcomprises both antigen-binding sites. Accordingly, the antibody can bethe Fab or F(ab′)₂. The Fab can include the heavy chain polypeptide andthe light chain polypeptide. The heavy chain polypeptide of the Fab caninclude the VH region and the CH1 region. The light chain of the Fab caninclude the VL region and CL region.

The antibody can be a polyclonal or monoclonal antibody. The antibodycan be a chimeric antibody, a single chain antibody, an affinity maturedantibody, a human antibody, a humanized antibody, or a fully humanantibody. The humanized antibody can be an antibody from a non-humanspecies that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule.

b. Antigen

The vaccine can also comprise an antigen, or fragment or variantthereof. The antigen can be anything that induces an immune response ina subject. The antigen can be a nucleic acid sequence, an amino acidsequence, or a combination thereof. The nucleic acid sequence can beDNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combinationthereof. The nucleic acid sequence can also include additional sequencesthat encode linker or tag sequences that are linked to the antigen by apeptide bond. The amino acid sequence can be a protein, a peptide, avariant thereof, a fragment thereof, or a combination thereof.

The antigen can be contained in a protein, a nucleic acid, or a fragmentthereof, or a variant thereof, or a combination thereof from any numberof organisms, for example, a virus, a parasite, a bacterium, a fungus,or a mammal. The antigen can be associated with an autoimmune disease,allergy, or asthma. In other embodiments, the antigen can be associatedwith cancer, herpes, influenza, hepatitis B, hepatitis C, humanpapilloma virus (HPV), or human immunodeficiency virus (HIV).Preferably, the antigen can be associated with influenza or HIV.

Some antigens can induce a strong immune response. Other antigens caninduce a weak immune response. The antigen can elicit a greater immuneresponse when combined with the PD1 antibody or PDL1 antibody asdescribed above.

(1) Viral Antigens

The antigen can be a viral antigen, or fragment thereof, or variantthereof. The viral antigen can be from a virus from one of the followingfamilies: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae,Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae,Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae,Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, orTogaviridae. The viral antigen can be from papilloma viruses, forexample, human papillomoa virus (HPV), human immunodeficiency virus(HIV), polio virus, hepatitis viruses, for example, hepatitis A virus(HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis Dvirus (HDV), and hepatitis E virus (HEV), smallpox virus (Variola majorand minor), vaccinia virus, influenza virus, rhinoviruses, dengue fevervirus, equine encephalitis viruses, rubella virus, yellow fever virus,Norwalk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-I),hairy cell leukemia virus (HTLV-II), California encephalitis virus,Hanta virus (hemorrhagic fever), rabies virus, Ebola fever virus,Marburg virus, measles virus, mumps virus, respiratory syncytial virus(RSV), herpes simplex 1 (oral herpes), herpes simplex 2 (genitalherpes), herpes zoster (varicella-zoster, a.k.a., chickenpox),cytomegalovirus (CMV), for example human CMV, Epstein-Barr virus (EBV),flavivirus, foot and mouth disease virus, chikungunya virus, lassavirus, arenavirus, or cancer causing virus.

(a) Hepatitis Antigen

The checkpoint inhibitors can be associated or combined with a hepatitisvirus antigen (i.e., hepatitis antigen), or fragment thereof, or variantthereof. The hepatitis antigen can be an antigen or immunogen fromhepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus(HCV), hepatitis D virus (HDV), and/or hepatitis E virus (HEV). In someembodiments, the hepatitis antigen can be a nucleic acid molecule(s),such as a plasmid(s), which encodes one or more of the antigens fromHAV, HBV, HCV, HDV, and HEV. The hepatitis antigen can be full-length orimmunogenic fragments of full-length proteins.

The hepatitis antigen can comprise consensus sequences and/ormodification for improved expression. Genetic modifications includingcodon optimization, RNA optimization, and the addition of a highefficient immunoglobulin leader sequence to increase the immunogenicityof the constructs can be included in the modified consensus sequences.The consensus hepatitis antigen may comprise a signal peptide such as animmunoglobulin signal peptide such as an IgE or IgG signal peptide, andin some embodiments, may comprise an HA tag. The immunogens can bedesigned to elicit stronger and broader cellular immune responses thancorresponding codong optimized immunogens.

The hepatitis antigen can be an antigen from HAV. The hepatitis antigencan be a HAV capsid protein, a HAV non-structural protein, a fragmentthereof, a variant thereof, or a combination thereof.

The hepatitis antigen can be an antigen from HCV. The hepatitis antigencan be a HCV nucleocapsid protein (i.e., core protein), a HCV envelopeprotein (e.g., E1 and E2), a HCV non-structural protein (e.g., NS1, NS2,NS3, NS4a, NS4b, NS5a, and NS5b), a fragment thereof, a variant thereof,or a combination thereof.

The hepatitis antigen can be an antigen from HDV. The hepatitis antigencan be a HDV delta antigen, fragment thereof, or variant thereof.

The hepatitis antigen can be an antigen from HEV. The hepatitis antigencan be a HEV capsid protein, fragment thereof, or variant thereof.

The hepatitis antigen can be an antigen from HBV. The hepatitis antigencan be a HBV core protein, a HBV surface protein, a HBV DNA polymerase,a HBV protein encoded by gene X, fragment thereof, variant thereof, orcombination thereof. The hepatitis antigen can be a HBV genotype A coreprotein, a HBV genotype B core protein, a HBV genotype C core protein, aHBV genotype D core protein, a HBV genotype E core protein, a HBVgenotype F core protein, a HBV genotype G core protein, a HBV genotype Hcore protein, a HBV genotype A surface protein, a HBV genotype B surfaceprotein, a HBV genotype C surface protein, a HBV genotype D surfaceprotein, a HBV genotype E surface protein, a HBV genotype F surfaceprotein, a HBV genotype G surface protein, a HBV genotype H surfaceprotein, fragment thereof, variant thereof, or combination thereof. Thehepatitis antigen can be a consensus HBV core protein, or a consensusHBV surface protein.

In some embodiments, the hepatitis antigen can be a HBV genotype Aconsensus core DNA sequence construct, an IgE leader sequence linked toa consensus sequence for HBV genotype A core protein, or a HBV genotypeA consensus core protein sequence.

In other embodiments, the hepatitis antigen can be a HBV genotype Bconsensus core DNA sequence construct, an IgE leader sequence linked toa consensus sequence for HBV genotype B core protein, or a HBV genotypeB consensus core protein sequence.

In still other embodiments, the hepatitis antigen can be a HBV genotypeC consensus core DNA sequence construct, an IgE leader sequence linkedto a consensus sequence for HBV genotype C core protein, or a HBVgenotype C consensus core protein sequence.

In some embodiments, the hepatitis antigen can be a HBV genotype Dconsensus core DNA sequence construct, an IgE leader sequence linked toa consensus sequence for HBV genotype D core protein, or a HBV genotypeD consensus core protein sequence.

In other embodiments, the hepatitis antigen can be a HBV genotype Econsensus core DNA sequence construct, an IgE leader sequence linked toa consensus sequence for HBV genotype E core protein, or a HBV genotypeE consensus core protein sequence.

In some embodiments, the hepatitis antigen can be a HBV genotype Fconsensus core DNA sequence construct, an IgE leader sequence linked toa consensus sequence for HBV genotype F core protein, or a HBV genotypeF consensus core protein sequence.

In other embodiments, the hepatitis antigen can be a HBV genotype Gconsensus core DNA sequence construct, an IgE leader sequence linked toa consensus sequence for HBV genotype G core protein, or a HBV genotypeG consensus core protein sequence.

In some embodiments, the hepatitis antigen can be a HBV genotype Hconsensus core DNA sequence construct, an IgE leader sequence linked toa consensus sequence for HBV genotype H core protein, or a HBV genotypeH consensus core protein sequence.

In still other embodiments, the hepatitis antigen can be a HBV genotypeA consensus surface DNA sequence construct, an IgE leader sequencelinked to a consensus sequence for HBV genotype A surface protein, or aHBV genotype A consensus surface protein sequence.

In some embodiments, the hepatitis antigen can be a HBV genotype Bconsensus surface DNA sequence construct, an IgE leader sequence linkedto a consensus sequence for HBV genotype B surface protein, or a HBVgenotype B consensus surface protein sequence.

In other embodiments, the hepatitis antigen can be a HBV genotype Cconsensus surface DNA sequence construct, an IgE leader sequence linkedto a consensus sequence for HBV genotype C surface protein, or a HBVgenotype C consensus surface protein sequence.

In still other embodiments, the hepatitis antigen can be a HBV genotypeD consensus surface DNA sequence construct, an IgE leader sequencelinked to a consensus sequence for HBV genotype D surface protein, or aHBV genotype D consensus surface protein sequence.

In some embodiments, the hepatitis antigen can be a HBV genotype Econsensus surface DNA sequence construct, an IgE leader sequence linkedto a consensus sequence for HBV genotype E surface protein, or a HBVgenotype E consensus surface protein sequence.

In other embodiments, the hepatitis antigen can be a HBV genotype Fconsensus surface DNA sequence construct, an IgE leader sequence linkedto a consensus sequence for HBV genotype F surface protein, or a HBVgenotype F consensus surface protein sequence.

In still other embodiments, the hepatitis antigen can be a HBV genotypeG consensus surface DNA sequence construct, an IgE leader sequencelinked to a consensus sequence for HBV genotype G surface protein, or aHBV genotype G consensus surface protein sequence.

In other embodiments, the hepatitis antigen can be a HBV genotype Hconsensus surface DNA sequence construct, an IgE leader sequence linkedto a consensus sequence for HBV genotype H surface protein, or a HBVgenotype H consensus surface protein sequence.

(b) Human Papilloma Virus (HPV) Antigen

checkpoint inhibitors can be associated or combined with a humanpapilloma virus (HPV) antigen, or fragment thereof, or variant thereof.The HPV antigen can be from HPV types 16, 18, 31, 33, 35, 45, 52, and 58which cause cervical cancer, rectal cancer, and/or other cancers. TheHPV antigen can be from HPV types 6 and 11, which cause genital warts,and are known to be causes of head and neck cancer.

The HPV antigens can be the HPV E6 or E7 domains from each HPV type. Forexample, for HPV type 16 (HPV16), the HPV16 antigen can include theHPV16 E6 antigen, the HPV16 E7 antigen, fragments, variants, orcombinations thereof. Similarly, the HPV antigen can be HPV 6 E6 and/orE7, HPV 11 E6 and/or E7, HPV 18 E6 and/or E7, HPV 31 E6 and/or E7, HPV33 E6 and/or E7, HPV 52 E6 and/or E7, or HPV 58 E6 and/or E7, fragments,variants, or combinations thereof.

(c) RSV Antigen

The checkpoint inhibitors can also be associated or combined with an RSVantigen or fragment thereof, or variant thereof. The RSV antigen can bea human RSV fusion protein (also referred to herein as “RSV F”, “RSV Fprotein” and “F protein”), or fragment or variant thereof. The human RSVfusion protein can be conserved between RSV subtypes A and B. The RSVantigen can be a RSV F protein, or fragment or variant thereof, from theRSV Long strain (GenBank AAX23994.1). The RSV antigen can be a RSV Fprotein from the RSV A2 strain (GenBank AAB59858.1), or a fragment orvariant thereof. The RSV antigen can be a monomer, a dimer or trimer ofthe RSV F protein, or a fragment or variant thereof. The RSV antigen canbe an optimized amino acid RSV F amino acid sequence, or fragment orvariant thereof.

The postfusion form of RSV F elicits high titer neutralizing antibodiesin immunized animals and protects the animals from RSV challenge. Thepresent invention utilizes this immunoresponse in the claimed vaccines.According to the invention, the RSV F protein can be in a prefusion formor a postfusion form.

The RSV antigen can also be human RSV attachment glycoprotein (alsoreferred to herein as “RSV G”, “RSV G protein” and “G protein”), orfragment or variant thereof. The human RSV G protein differs between RSVsubtypes A and B. The antigen can be RSV G protein, or fragment orvariant thereof, from the RSV Long strain (GenBank AAX23993). The RSVantigen can be RSV G protein from: the RSV subtype B isolate H5601, theRSV subtype B isolate H1068, the RSV subtype B isolate H5598, the RSVsubtype B isolate H1123, or a fragment or variant thereof. The RSVantigen can be an optimized amino acid RSV G amino acid sequence, orfragment or variant thereof.

In other embodiments, the RSV antigen can be human RSV non-structuralprotein 1 (“NS1 protein”), or fragment or variant thereof. For example,the RSV antigen can be RSV NS1 protein, or fragment or variant thereof,from the RSV Long strain (GenBank AAX23987.1). The RSV antigen human canalso be RSV non-structural protein 2 (“NS2 protein”), or fragment orvariant thereof. For example, the RSV antigen can be RSV NS2 protein, orfragment or variant thereof, from the RSV Long strain (GenBankAAX23988.1). The RSV antigen can further be human RSV nucleocapsid (“N”)protein, or fragment or variant thereof. For example, the RSV antigencan be RSV N protein, or fragment or variant thereof, from the RSV Longstrain (GenBank AAX23989.1). The RSV antigen can be human RSVPhosphoprotein (“P”) protein, or fragment or variant thereof. Forexample, the RSV antigen can be RSV P protein, or fragment or variantthereof, from the RSV Long strain (GenBank AAX23990.1). The RSV antigenalso can be human RSV Matrix protein (“M”) protein, or fragment orvariant thereof. For example, the RSV antigen can be RSV M protein, orfragment or variant thereof, from the RSV Long strain (GenBankAAX23991.1).

In still other embodiments, the RSV antigen can be human RSV smallhydrophobic (“SH”) protein, or fragment or variant thereof. For example,the RSV antigen can be RSV SH protein, or fragment or variant thereof,from the RSV Long strain (GenBank AAX23992.1). The RSV antigen can alsobe human RSV Matrix protein 2-1 (“M2-1”) protein, or fragment or variantthereof. For example, the RSV antigen can be RSV M2-1 protein, orfragment or variant thereof, from the RSV Long strain (GenBankAAX23995.1). The RSV antigen can further be human RSV Matrix protein 2-2(“M2-2”) protein, or fragment or variant thereof. For example, the RSVantigen can be RSV M2-2 protein, or fragment or variant thereof, fromthe RSV Long strain (GenBank AAX23997.1). The RSV antigen human can beRSV Polymerase L (“L”) protein, or fragment or variant thereof. Forexample, the RSV antigen can be RSV L protein, or fragment or variantthereof, from the RSV Long strain (GenBank AAX23996.1).

In further embodiments, the RSV antigen can have an optimized amino acidsequence of NS1, NS2, N, P, M, SH, M2-1, M2-2, or L protein. The RSVantigen can be a human RSV protein or recombinant antigen, such as anyone of the proteins encoded by the human RSV genome.

In other embodiments, the RSV antigen can be, but is not limited to, theRSV F protein from the RSV Long strain, the RSV G protein from the RSVLong strain, the optimized amino acid RSV G amino acid sequence, thehuman RSV genome of the RSV Long strain, the optimized amino acid RSV Famino acid sequence, the RSV NS1 protein from the RSV Long strain, theRSV NS2 protein from the RSV Long strain, the RSV N protein from the RSVLong strain, the RSV P protein from the RSV Long strain, the RSV Mprotein from the RSV Long strain, the RSV SH protein from the RSV Longstrain, the RSV M2-1 protein from the RSV Long strain, the RSV M2-2protein from the RSV Long strain, the RSV L protein from the RSV Longstrain, the RSV G protein from the RSV subtype B isolate H5601, the RSVG protein from the RSV subtype B isolate H1068, the RSV G protein fromthe RSV subtype B isolate H5598, the RSV G protein from the RSV subtypeB isolate H1123, or fragment thereof, or variant thereof.

(d) Influenza Antigen

The checkpoint inhibitors can be associated or combined with aninfluenza antigen or fragment thereof, or variant thereof. The influenzaantigens are those capable of eliciting an immune response in a mammalagainst one or more influenza serotypes. The antigen can comprise thefull length translation product HA0, subunit HA1, subunit HA2, a variantthereof, a fragment thereof or a combination thereof. The influenzahemagglutinin antigen can be a consensus sequence derived from multiplestrains of influenza A serotype H1, a consensus sequence derived frommultiple strains of influenza A serotype H2, a hybrid sequencecontaining portions of two different consensus sequences derived fromdifferent sets of multiple strains of influenza A serotype H1 or aconsensus sequence derived from multiple strains of influenza B. Theinfluenza hemagglutinin antigen can be from influenza B.

The influenza antigen can also contain at least one antigenic epitopethat can be effective against particular influenza immunogens againstwhich an immune response can be induced. The antigen may provide anentire repertoire of immunogenic sites and epitopes present in an intactinfluenza virus. The antigen may be a consensus hemagglutinin antigensequence that can be derived from hemagglutinin antigen sequences from aplurality of influenza A virus strains of one serotype such as aplurality of influenza A virus strains of serotype H1 or of serotype H2.The antigen may be a hybrid consensus hemagglutinin antigen sequencethat can be derived from combining two different consensus hemagglutininantigen sequences or portions thereof. Each of two different consensushemagglutinin antigen sequences may be derived from a different set of aplurality of influenza A virus strains of one serotype such as aplurality of influenza A virus strains of serotype H1. The antigen maybe a consensus hemagglutinin antigen sequence that can be derived fromhemagglutinin antigen sequences from a plurality of influenza B virusstrains.

In some embodiments, the influenza antigen can be H1 HA, H2 HA, H3 HA,H5 HA, or a BHA antigen. Alternatively, the influenza antigen can be aconsensus hemagglutinin antigen comprising a consensus H1 amino acidsequence or a consensus H2 amino acid sequence. The consensushemagglutinin antigen may be a synthetic hybrid consensus H1 sequencecomprising portions of two different consensus H1 sequences, which areeach derived from a different set of sequences from the other. Anexample of a consensus HA antigen that is a synthetic hybrid consensusH1 protein is a protein comprising the U2 amino acid sequence. Theconsensus hemagglutinin antigen may be a consensus hemagglutinin proteinderived from hemagglutinin sequences from influenza B strains, such as aprotein comprising the consensus BHA amino acid sequence.

The consensus hemagglutinin antigen may further comprise one or moreadditional amino acid sequence elements. The consensus hemagglutininantigen may further comprise on its N-terminal an IgE or IgG leaderamino acid sequence.The consensus hemagglutinin antigen may furthercomprise an immunogenic tag which is a unique immunogenic epitope thatcan be detected by readily available antibodies. An example of such animmunogenic tag is the 9 amino acid influenza HA Tag which may be linkedon the consensus hemagglutinin C terminus. In some embodiments,consensus hemagglutinin antigen may further comprise on its N-terminalan IgE or IgG leader amino acid sequence and on its C terminal an HAtag.

The consensus hemagglutinin antigen may be a consensus hemagglutininprotein that consists of consensus influenza amino acid sequences orfragments and variants thereof. The consensus hemagglutinin antigen maybe a consensus hemagglutinin protein that comprises non-influenzaprotein sequences and influenza protein sequences or fragments andvariants thereof. Examples of a consensus H1 protein include those thatmay consist of the consensus H1 amino acid sequence or those thatfurther comprise additional elements such as an IgE leader sequence, oran HA Tag or both an IgE leader sequence and an HA Tag.

Examples of consensus H2 proteins include those that may consist of theconsensus H2 amino acid sequence or those that further comprise an IgEleader sequence, or an HA Tag, or both an IgE leader sequence and an HATag.

Examples of hybrid consensus H1 proteins include those that may consistof the consensus U2 amino acid sequence or those that further comprisean IgE leader sequence, or an HA Tag, or both an IgE leader sequence andan HA Tag.

Examples of hybrid consensus influenza B hemagglutinin proteins includethose that may consist of the consensus BHA amino acid sequence or itmay comprise an IgE leader sequence, or an HA Tag, or both an IgE leadersequence and an HA Tag.

The consensus hemagglutinin protein can be encoded by a consensushemagglutinin nucleic acid, a variant thereof or a fragment thereof.Unlike the consensus hemagglutinin protein which may be a consensussequence derived from a plurality of different hemagglutinin sequencesfrom different strains and variants, the consensus hemagglutinin nucleicacid refers to a nucleic acid sequence that encodes a consensus proteinsequence and the coding sequences used may differ from those used toencode the particular amino acid sequences in the plurality of differenthemagglutinin sequences from which the consensus hemagglutinin proteinsequence is derived. The consensus nucleic acid sequence may be codonoptimized and/or RNA optimized. The consensus hemagglutinin nucleic acidsequence may comprise a Kozak's sequence in the 5′ untranslated region.The consensus hemagglutinin nucleic acid sequence may comprise nucleicacid sequences that encode a leader sequence. The coding sequence of anN terminal leader sequence is 5′ of the hemagglutinin coding sequence.The N-terminal leader can facilitate secretion. The N-terminal leadercan be an IgE leader or an IgG leader. The consensus hemagglutininnucleic acid sequence can comprise nucleic acid sequences that encode animmunogenic tag. The immunogenic tag can be on the C terminus of theprotein and the sequence encoding it is 3′ of the HA coding sequence.The immunogenic tag provides a unique epitope for which there arereadily available antibodies so that such antibodies can be used inassays to detect and confirm expression of the protein. The immunogenictag can be an H Tag at the C-terminus of the protein.

(e) Human Immunodeficiency Virus (HIV) Antigen

The checkpoint inhibitors can be associated or combined with an HIVantigen or fragment thereof, or variant thereof. HIV antigens caninclude modified consensus sequences for immunogens. Geneticmodifications including codon optimization, RNA optimization, and theaddition of a high efficient immunoglobin leader sequence to increasethe immunogenicity of constructs can be included in the modifiedconsensus sequences. The novel immunogens can be designed to elicitstronger and broader cellular immune responses than a correspondingcodon optimized immunogens.

In some embodiments, the HIV antigen can be a subtype A consensusenvelope DNA sequence construct, an IgE leader sequence linked to aconsensus sequence for Subtype A envelope protein, or a subtype Aconsensus Envelope protein sequence.

In other embodiments, the HIV antigen can be a subtype B consensusenvelope DNA sequence construct, an IgE leader sequence linked to aconsensus sequence for Subtype B envelope protein, or an subtype Bconsensus Envelope protein sequence.

In still other embodiments, the HIV antigen can be a subtype C consensusenvelope DNA sequence construct, an IgE leader sequence linked to aconsensus sequence for subtype C envelope protein, or a subtype Cconsensus envelope protein sequence.

In further embodiments, the HIV antigen can be a subtype D consensusenvelope DNA sequence construct, an IgE leader sequence linked to aconsensus sequence for Subtype D envelope protein, or a subtype Dconsensus envelope protein sequence.

In some embodiments, the HIV antigen can be a subtype B Nef-Revconsensus envelope DNA sequence construct, an IgE leader sequence linkedto a consensus sequence for Subtype B Nef-Rev protein, or a Subtype BNef-Rev consensus protein sequence.

In other embodiments, the HIV antigen can be a Gag consensus DNAsequence of subtype A, B, C and D DNA sequence construct, an IgE leadersequence linked to a consensus sequence for Gag consensus subtype A, B,C and D protein, or a consensus Gag subtype A, B, C and D proteinsequence.

In still other embodiments the HIV antigen can be a MPol DNA sequence ora MPol protein sequence. The HIV antigen can be nucleic acid or aminoacid sequences of Env A, Env B, Env C, Env D, B Nef-Rev, Gag, or anycombination thereof.

(f) Herpes Antigens Including HCMV, HSV1, HSV2, CEHV1, and VZV

The herpes antigens comprise immunogenic proteins including gB, gM, gN,gH, gL, gO, gE, gI, gK, gC, gD, UL128, UL130, UL-131A, UL-83 (pp65),whether from HCMV, HSV1, HSV2, CeHV1, or VZV. In some embodiments, theantigens can be HSV1-gH, HSV1-gL, HSV1-gC, HSV1-gD, HSV2-gH, HSV2-gL,HSV2-gC, HSV2-gD, VZV-gH, VZV-gL, VZV-gM, VZV-gN, CeHV1-gH, CeHV1-gL,CeHV1-gC, CeHV1-gD, VZV-gE, or VZV-gl.

(2) Parasite Antigens

The antigen can be a parasite antigen or fragment or variant thereof.The parasite can be a protozoa, helminth, or ectoparasite. The helminth(i.e., worm) can be a flatworm (e.g., flukes and tapeworms), athorny-headed worm, or a round worm (e.g., pinworms). The ectoparasitecan be lice, fleas, ticks, and mites.

The parasite can be any parasite causing the following diseases:Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis,Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis,Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis,Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis,Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis,Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lymedisease, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis,Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis,Taeniasis, Toxocariasis, Toxoplasmosis, Trichinosis, and Trichuriasis.

The parasite can be Acanthamoeba, Anisakis, Ascaris lumbricoides,Botfly, Balantidium coli, Bedbug, Cestoda (tapeworm), Chiggers,Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica,Giardia lamblia, Hookworm, Leishmania, Linguatula serrata, Liver fluke,Loa loa, Paragonimus—lung fluke, Pinworm, Plasmodium falciparum,Schistosoma, Strongyloides stercoralis, Mite, Tapeworm, Toxoplasmagondii, Trypanosoma, Whipworm, or Wuchereria bancrofti.

(a) Malaria Antigen

The checkpoint inhibitors can be associated or combined with a malariaantigen (i.e., PF antigen or PF immunogen), or fragment thereof, orvariant thereof. The antigen can be from a parasite causing malaria. Themalaria causing parasite can be Plasmodium falciparum. The Plasmodiumfalciparum antigen can include the circumsporozoite (CS) antigen.

In some embodiments, the malaria antigen can be nucleic acid moleculessuch as plasmids which encode one or more of the P. falciparumimmunogens CS; LSA1; TRAP; CelTOS; and Ama1. The immunogens may be fulllength or immunogenic fragments of full length proteins. The immunogenscomprise consensus sequences and/or modifications for improvedexpression.

In other embodiments, the malaria antigen can be a consensus sequence ofTRAP, which is also referred to as SSP2, designed from a compilation ofall full-length Plasmodium falciparum TRAP/SSP2 sequences in the GenBankdatabase (28 sequences total). Consensus TRAP immunogens (i.e., ConTRAPimmunogen) may comprise a signal peptide such as an immunoglobulinsignal peptide such as an IgE or IgG signal peptide and in someembodiments, may comprise an HA Tag.

In still other embodiments, the malaria antigen can be CelTOS, which isalso referred to as Ag2 and is a highly conserved Plasmodium antigen.Consensus CelTOS antigens (i.e., ConCelTOS immunogen) may comprise asignal peptide such as an immunoglobulin signal peptide such as an IgEor IgG signal peptide and in some embodiments, may comprise an HA Tag.

In further embodiments, the malaria antigen can be Ama1, which is ahighly conserved Plasmodium antigen. The malaria antigen can also be aconsensus sequence of Ama1 (i.e., ConAma1 immunogen) comprising in someinstances, a signal peptide such as an immunoglobulin signal peptidesuch as an IgE or IgG signal peptide and in some embodiments, maycomprise an HA Tag.

In some embodiments, the malaria antigen can be a consensus CS antigen(i.e., Consensus CS immunogen) comprising in some instances, a signalpeptide such as an immunoglobulin signal peptide such as an IgE or IgGsignal peptide and in some embodiments, may comprise an HA Tag.

In other embodiments, the malaria antigen can be a fusion proteincomprising a combination of two or more of the PF proteins set forthherein. For example, fusion proteins may comprise two or more ofConsensus CS immunogen, ConLSA1 immunogen, ConTRAP immunogen, ConCelTOSimmunogen and ConAma1 immunogen linked directly adjacent to each otheror linked with a spacer or one or more amino acids in between. In someembodiments, the fusion protein comprises two PF immunogens; in someembodiments the fusion protein comprises three PF immunogens, in someembodiments the fusion protein comprises four PF immunogens, and in someembodiments the fusion protein comprises five PF immunogens. Fusionproteins with two Consensus PF immunogens may comprise: CS and LSA1; CSand TRAP; CS and CelTOS; CS and Ama1; LSA1 and TRAP; LSA1 and CelTOS;LSA1 and Ama1; TRAP and CelTOS; TRAP and Ama1; or CelTOS and Ama1.Fusion proteins with three Consensus PF immunogens may comprise: CS,LSA1 and TRAP; CS, LSA1 and CelTOS; CS, LSA1 and Ama1; LSA1, TRAP andCelTOS; LSA1, TRAP and Ama1; or TRAP, CelTOS and Ama1. Fusion proteinswith four Consensus PF immunogens may comprise: CS, LSA1, TRAP andCelTOS; CS, LSA1, TRAP and Ama1; CS, LSA1, CelTOS and Ama1; CS, TRAP,CelTOS and Ama1; or LSA1, TRAP, CelTOS and Ama1. Fusion proteins withfive Consensus PF immunogens may comprise CS or CS-alt, LSA1, TRAP,CelTOS and Ama1.

In some embodiments, the fusion proteins comprise a signal peptidelinked to the N terminus. In some embodiments, the fusion proteinscomprise multiple signal peptides linked to the N terminal of eachConsensus PF immunogen. In some embodiments, a spacer may be includedbetween PF immunogens of a fusion protein. In some embodiments, thespacer between PF immunogens of a fusion protein may be a proteolyiccleavage site. In some embodiments, the spacer may be a proteolyiccleavage site recognized by a protease found in cells to which thevaccine is intended to be administered and/or taken up. In someembodiments, a spacer may be included between PF immunogens of a fusionprotein wherein the spacer is a proteolyic cleavage site recognized by aprotease found in cells to which the vaccine is intended to beadministered and/or taken up and the fusion proteins comprises multiplesignal peptides linked to the N terminal of each Consensus PF immunogenssuch that upon cleavage the signal peptide of each Consensus PFimmunogens translocates the Consensus PF immunogen to outside the cell.

(3) Bacterial Antigens

The antigen can be a bacterial antigen or fragment or variant thereof.The bacterium can be from any one of the following phyla: Acidobacteria,Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae,Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres,Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres,Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira,Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes,Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

The bacterium can be a gram positive bacterium or a gram negativebacterium. The bacterium can be an aerobic bacterium or an anerobicbacterium. The bacterium can be an autotrophic bacterium or aheterotrophic bacterium. The bacterium can be a mesophile, aneutrophile, an extremophile, an acidophile, an alkaliphile, athermophile, a psychrophile, an halophile, or an osmophile.

The bacterium can be an anthrax bacterium, an antibiotic resistantbacterium, a disease causing bacterium, a food poisoning bacterium, aninfectious bacterium, Salmonella bacterium, Staphylococcus bacterium,Streptococcus bacterium, or tetanus bacterium. The bacterium can be amycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthraces,methicillin-resistant Staphylococcus aureus (MRSA), or Clostridiumdifficile. The bacterium can be Mycobacterium tuberculosis.

(a) Mycobacterium Tuberculosis Antigens

The checkpoint inhibitors can be associated or combined with aMycobacterium tuberculosis antigen (i.e., TB antigen or TB immunogen),or fragment thereof, or variant thereof. The TB antigen can be from theAg85 family of TB antigens, for example, Ag85A and Ag85B. The TB antigencan be from the Esx family of TB antigens, for example, EsxA, EsxB,EsxC, EsxD, EsxE, EsxF, EsxH, EsxO, EsxQ, EsxR, EsxS, EsxT, EsxU, EsxV,and EsxW.

In some embodiments, the TB antigen can be nucleic acid molecules suchas plasmids which encode one or more of the Mycobacterium tuberculosisimmunogens from the Ag85 family and the Esx family. The immunogens canbe full-length or immunogenic fragments of full-length proteins. Theimmunogens can comprise consensus sequences and/or modifications forimproved expression. Consensus immunogens may comprise a signal peptidesuch as an immunoglobulin signal peptide such as an IgE or IgG signalpeptide and in some embodiments, may comprise an HA tag.

(4) Fungal Antigens

The antigen can be a fungal antigen or fragment or variant thereof. Thefungus can be Aspergillus species, Blastomyces dermatitides, Candidayeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans,Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasmacapsulatum, Mucoromycotina, Pneumocystis provecii, Sporothrix schenckii,Exserohilum, or Cladosporium.

(5) Cancer Markers

Markers are known proteins that are present or unregulated vis-à-viscertain cancer cells. By methodology of generating antigens thatrepresent such markers in a way to break tolerance to self, a cancervaccine can be generated. Such cancer vaccines can include thecheckpoint inhibitors to enhance the immune response. The following aresome cancer antigens:

a. hTERT

hTERT is a human telomerase reverse transcriptase that synthesizes aTTAGGG tag on the end of telomeres to prevent cell death due tochromosomal shortening. Hyperproliferative cells with abnormally highexpression of hTERT may be targeted by immunotherapy. Recent studiesdemonstrate that hTERT expression in dendritic cells transfected withhTERT genes can induce CD8+ cytotoxic T cells and elicit a CD4+ T cellsin an antigen-specific fashion.

hTERT can be administered in vectors described herein, and combined withcheckpoint inhibitors in various vaccination schedules, including thatin the Example, below.

b. Prostate Antigens

The following are antigens capable of eliciting an immune response in amammal against a prostate antigen. The consensus antigen can compriseepitopes that make them particularly effective as immunogens againstprostate cancer cells can be induced. The consensus prostate antigen cancomprise the full length translation product, a variant thereof, afragment thereof or a combination thereof.

The prostate antigens can include one or more of the following: PSAantigen, PSMA antigen, STEAP antigen, PSCA antigen, Prostatic acidphosphatase (PAP) antigen, and other known prostate cancer markers.Proteins may comprise sequences homologous to the prostate antigens,fragments of the prostate antigens and proteins with sequenceshomologous to fragments of the prostate antigens.

The prostate antigens can be administered in vectors described herein,and combined with checkpoint inhibitors in various vaccinationschedules, including that in the Example, below.

c. WT1

The antigen can be Wilm's tumor suppressor gene 1 (WT1), a fragmentthereof, a variant thereof, or a combination thereof. WT1 is atranscription factor containing at the N-terminus, aproline/glutamine-rich DNA-binding domain and at the C-terminus, fourzinc finger motifs. WT1 plays a role in the normal development of theurogenital system and interacts with numerous factors, for example, p53,a known tumor suppressor and the serine protease HtrA2, which cleavesWT1 at multiple sites after treatment with a cytotoxic drug.

Mutation of WT1 can lead to tumor or cancer formation, for example,Wilm's tumor or tumors expressing WT1. Wilm's tumor often forms in oneor both kidneys before metastasizing to other tissues, for example, butnot limited to, liver tissue, urinary tract system tissue, lymph tissue,and lung tissue. Accordingly, Wilm's tumor can be considered ametastatic tumor. Wilm's tumor usually occurs in younger children (e.g.,less than 5 years old) and in both sporadic and hereditary forms.Accordingly, the vaccine can be used for treating subjects sufferingfrom Wilm's tumor. The vaccine can also be used for treating subjectswith cancers or tumors that express WT1for preventing development ofsuch tumors in subjects. The WT1 antigen can differ from the native,“normal” WT1 gene, and thus, provide therapy or prophylaxis against anWT1 antigen-expressing tumor. Proteins may comprise sequences homologousto the WT1 antigens, fragments of the WT1 antigens and proteins withsequences homologous to fragments of the WT1 antigens.

The WT1 antigens can be administered in vectors described herein, andcombined with checkpoint inhibitors in various vaccination schedules,including that in the Example, below.

d. Tyrosinase Antigen

The antigen tyrosinase (Tyr) antigen is an important target for immunemediated clearance by inducing (1) humoral immunity via B cell responsesto generate antibodies that block monocyte chemoattractant protein-1(MCP-1) production, thereby retarding myeloid derived suppressor cells(MDSCs) and suppressing tumor growth; (2) increase cytotoxic Tlymphocyte such as CD8⁺ (CTL) to attack and kill tumor cells; (3)increase T helper cell responses; (4) and increase inflammatoryresponses via IFN-γ and TFN-α or preferably all of the aforementioned.

Tyrosinase is a copper-containing enzyme that can be found in plant andanimal tissues. Tyrosinase catalyzes the production of melanin and otherpigments by the oxidation of phenols such as tyrosine. In melanoma,tyrosinase can become unregulated, resulting in increased melaninsynthesis. Tyrosinase is also a target of cytotoxic T cell recognitionin subjects suffering from melanoma. Accordingly, tyrosinase can be anantigen associated with melanoma.

The antigen can comprise protein epitopes that make them particularlyeffective as immunogens against which anti-Tyr immune responses can beinduced. The Tyr antigen can comprise the full length translationproduct, a variant thereof, a fragment thereof or a combination thereof.

The Tyr antigen can comprise a consensus protein. The Tyr antigeninduces antigen-specific T-cell and high titer antibody responses bothsystemically against all cancer and tumor related cells. As such, aprotective immune response is provided against tumor formation byvaccines comprising the Tyr consensus antigen. Accordingly, any user candesign a vaccine of the present invention to include a Tyr antigen toprovide broad immunity against tumor formation, metastasis of tumors,and tumor growth. Proteins may comprise sequences homologous to the Tyrantigens, fragments of the Tyr antigens and proteins with sequenceshomologous to fragments of the Tyr antigens.

The Tyr antigens can be administered in vectors described herein, andcombined with checkpoint inhibitors in various vaccination schedules,including that in the Example, below.

e. NYES01

NY-ESO-1 is a cancer-testis antigen expressed in various cancers whereit can induce both cellular and humoral immunity. Gene expressionstudies have shown upregulation of the gene for NY-ESO-1, CTAG1B, inmyxoid and round cell liposarcomas. Proteins may comprise sequenceshomologous to the NYES01 antigens, fragments of the NYES01 antigens andproteins with sequences homologous to fragments of the NYES01 antigens.

The NYES01 antigens can be administered in vectors described herein, andcombined with checkpoint inhibitors in various vaccination schedules,including that in the Example, below.

f. PRAME

Melanoma antigen preferentially expressed in tumors (PRAME antigen) is aprotein that in humans is encoded by the PRAME gene. This gene encodesan antigen that is predominantly expressed in human melanomas and thatis recognized by cytolytic T lymphocytes. It is not expressed in normaltissues, except testis. The gene is also expressed in acute leukemias.Five alternatively spliced transcript variants encoding the same proteinhave been observed for this gene. Proteins may comprise sequenceshomologous to the PRAME antigens, fragments of the PRAME antigens andproteins with sequences homologous to fragments of the PRAME antigens.

The PRAME antigens can be administered in vectors described herein, andcombined with checkpoint inhibitors in various vaccination schedules,including that in the Example, below.

g. MAGE

MAGE stands for Melanoma-associated Antigen, and in particular melanomaassociated antigen 4 (MAGEA4). MAGE-A4 is expressed in male germ cellsand tumor cells of various histological types such as gastrointestinal,esophageal and pulmonary carcinomas. MAGE-A4 binds the oncoprotein,Gankyrin. This MAGE-A4 specific binding is mediated by its C-terminus.Studies have shown that exogenous MAGE-A4 can partly inhibit theadhesion-independent growth of Gankyrin-overexpressing cells in vitroand suppress the formation of migrated tumors from these cells in nudemice. This inhibition is dependent upon binding between MAGE-A4 andGankyrin, suggesting that interactions between Gankyrin and MAGE-A4inhibit Gankyrin-mediated carcinogenesis. It is likely that MAGEexpression in tumor tissue is not a cause, but a result of tumorgenesis, and MAGE genes take part in the immune process by targetingearly tumor cells for destruction.

Melanoma-associated antigen 4 protein (MAGEA4) can be involved inembryonic development and tumor transformation and/or progression.MAGEA4 is normally expressed in testes and placenta. MAGEA4, however,can be expressed in many different types of tumors, for example,melanoma, head and neck squamous cell carcinoma, lung carcinoma, andbreast carcinoma. Accordingly, MAGEA4 can be antigen associated with avariety of tumors.

The MAGEA4 antigen can induce antigen-specific T cell and/or high titerantibody responses, thereby inducing or eliciting an immune responsethat is directed to or reactive against the cancer or tumor expressingthe antigen. In some embodiments, the induced or elicited immuneresponse can be a cellular, humoral, or both cellular and humoral immuneresponses. In some embodiments, the induced or elicited cellular immuneresponse can include induction or secretion of interferon-gamma (IFN-γ)and/or tumor necrosis factor alpha (TNF-α). In other embodiments, theinduced or elicited immune response can reduce or inhibit one or moreimmune suppression factors that promote growth of the tumor or cancerexpressing the antigen, for example, but not limited to, factors thatdown regulate MHC presentation, factors that up regulateantigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines suchas IL-10 and TFG-β, tumor associated macrophages, tumor associatedfibroblasts.

The MAGEA4 antigen can comprise protein epitopes that make themparticularly effective as immunogens against which anti-MAGEA4 immuneresponses can be induced. The MAGEA4 antigen can comprise the fulllength translation product, a variant thereof, a fragment thereof or acombination thereof. The MAGEA4 antigen can comprise a consensusprotein.

The nucleic acid sequence encoding the consensus MAGEA4 antigen can beoptimized with regards to codon usage and corresponding RNA transcripts.The nucleic acid encoding the consensus MAGEA4 antigen can be codon andRNA optimized for expression. In some embodiments, the nucleic acidsequence encoding the consensus MAGEA4 antigen can include a Kozaksequence (e.g., GCC ACC) to increase the efficiency of translation. Thenucleic acid encoding the consensus MAGEA4 antigen can include multiplestop codons (e.g., TGA TGA) to increase the efficiency of translationtermination.

The MAGE antigens can be administered in vectors described herein, andcombined with checkpoint inhibitors in various vaccination schedules,including that in the Example, below.

c. Vector

The vaccine can comprise one or more vectors that include a nucleic acidencoding the antigen and the PD1 antibody or PDL1 antibody. The one ormore vectors can be capable of expressing the antigen and the PD1antibody or PDL1 antibody. The vector can have a nucleic acid sequencecontaining an origin of replication. The vector can be a plasmid,bacteriophage, bacterial artificial chromosome or yeast artificialchromosome. The vector can be either a self-replication extrachromosomal vector, or a vector which integrates into a host genome.

The one or more vectors can be an expression construct, which isgenerally a plasmid that is used to introduce a specific gene into atarget cell. Once the expression vector is inside the cell, the proteinthat is encoded by the gene is produced by the cellular-transcriptionand translation machinery ribosomal complexes. The plasmid is frequentlyengineered to contain regulatory sequences that act as enhancer andpromoter regions and lead to efficient transcription of the gene carriedon the expression vector. The vectors of the present invention expresslarge amounts of stable messenger RNA, and therefore proteins.

The vectors may have expression signals such as a strong promoter, astrong termination codon, adjustment of the distance between thepromoter and the cloned gene, and the insertion of a transcriptiontermination sequence and a PTIS (portable translation initiationsequence).

(1) Expression Vectors

The vector can be a circular plasmid or a linear nucleic acid. Thecircular plasmid and linear nucleic acid are capable of directingexpression of a particular nucleotide sequence in an appropriate subjectcell. The vector can have a promoter operably linked to theantigen-encoding nucleotide sequence, or the adjuvant-encodingnucleotide sequence, which may be operably linked to terminationsignals. The vector can also contain sequences required for propertranslation of the nucleotide sequence. The vector comprising thenucleotide sequence of interest may be chimeric, meaning that at leastone of its components is heterologous with respect to at least one ofits other components. The expression of the nucleotide sequence in theexpression cassette may be under the control of a constitutive promoteror of an inducible promoter, which initiates transcription only when thehost cell is exposed to some particular external stimulus. In the caseof a multicellular organism, the promoter can also be specific to aparticular tissue or organ or stage of development.

(2) Circular and Linear Vectors

The vector may be circular plasmid, which may transform a target cell byintegration into the cellular genome or exist extrachromosomally (e.g.,autonomous replicating plasmid with an origin of replication).

The vector can be pVAX, pcDNA3.0, or provax, or any other expressionvector capable of expressing DNA encoding the antigen, or the adjuvantand enabling a cell to translate the sequence to an antigen that isrecognized by the immune system, or the adjuvant.

Also provided herein is a linear nucleic acid vaccine, or linearexpression cassette (“LEC”), that is capable of being efficientlydelivered to a subject via electroporation and expressing one or moredesired antigens, or one or more desired adjuvants. The LEC may be anylinear DNA devoid of any phosphate backbone. The DNA may encode one ormore antigens, or one or more adjuvants. The LEC may contain a promoter,an intron, a stop codon, and/or a polyadenylation signal. The expressionof the antigen, or the adjuvant may be controlled by the promoter. TheLEC may not contain any antibiotic resistance genes and/or a phosphatebackbone. The LEC may not contain other nucleic acid sequences unrelatedto the desired antigen gene expression, or the desired adjuvantexpression.

The LEC may be derived from any plasmid capable of being linearized. Theplasmid may be capable of expressing the antigen, or the PD1 antibody orPDL1 antibody. The plasmid may be capable of expressing the PD1 antibodyor PDL1 antibody. The plasmid can be pNP (Puerto Rico/34) or pM2 (NewCaledonia/99). The plasmid may be WLV009, pVAX, pcDNA3.0, or provax, orany other expression vector capable of expressing DNA encoding theantigen, or encoding the adjuvant, and enabling a cell to translate thesequence to an antigen that is recognized by the immune system, or theadjuvant.

The LEC can be perM2. The LEC can be perNP. perNP and perMR can bederived from pNP (Puerto Rico/34) and pM2 (New Caledonia/99),respectively.

(3) Promoter, Intron, Stop Codon, and Polyadenylation Signal

The vector may have a promoter. A promoter may be any promoter that iscapable of driving gene expression and regulating expression of theisolated nucleic acid. Such a promoter is a cis-acting sequence elementrequired for transcription via a DNA dependent RNA polymerase, whichtranscribes the antigen sequence, or the adjuvant sequence describedherein. Selection of the promoter used to direct expression of aheterologous nucleic acid depends on the particular application. Thepromoter may be positioned about the same distance from thetranscription start in the vector as it is from the transcription startsite in its natural setting. However, variation in this distance may beaccommodated without loss of promoter function.

The promoter may be operably linked to the nucleic acid sequenceencoding the antigen and signals required for efficient polyadenylationof the transcript, ribosome binding sites, and translation termination.The promoter may be operably linked to the nucleic acid sequenceencoding the adjuvant and signals required for efficient polyadenylationof the transcript, ribosome binding sites, and translation termination.

The promoter may be a CMV promoter, SV40 early promoter, SV40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or another promotershown effective for expression in eukaryotic cells.

The vector may include an enhancer and an intron with functional splicedonor and acceptor sites. The vector may contain a transcriptiontermination region downstream of the structural gene to provide forefficient termination. The termination region may be obtained from thesame gene as the promoter sequence or may be obtained from differentgenes.

d. Excipients and other Components of the Vaccine

The vaccine may further comprise a pharmaceutically acceptableexcipient. The pharmaceutically acceptable excipient can be functionalmolecules such as vehicles, adjuvants other than the PD1 antibody orPDL1 antibody, carriers, or diluents. The pharmaceutically acceptableexcipient can be a transfection facilitating agent, which can includesurface active agents, such as immune-stimulating complexes (ISCOMS),Freunds incomplete adjuvant, LPS analog including monophosphoryl lipidA, muramyl peptides, quinone analogs, vesicles such as squalene andsqualene, hyaluronic acid, lipids, liposomes, calcium ions, viralproteins, polyanions, polycations, or nanoparticles, or other knowntransfection facilitating agents.

The transfection facilitating agent is a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. The transfectionfacilitating agent is poly-L-glutamate, and the poly-L-glutamate may bepresent in the vaccine at a concentration less than 6 mg/ml. Thetransfection facilitating agent may also include surface active agentssuch as immune-stimulating complexes (ISCOMS), Freunds incompleteadjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides,quinone analogs and vesicles such as squalene and squalene, andhyaluronic acid may also be used administered in conjunction with thegenetic construct. The DNA plasmid vaccines may also include atransfection facilitating agent such as lipids, liposomes, includinglecithin liposomes or other liposomes known in the art, as aDNA-liposome mixture (see for example W09324640), calcium ions, viralproteins, polyanions, polycations, or nanoparticles, or other knowntransfection facilitating agents. The transfection facilitating agent isa polyanion, polycation, including poly-L-glutamate (LGS), or lipid.Concentration of the transfection agent in the vaccine is less than 4mg/ml, less than 2 mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, lessthan 0.500 mg/ml, less than 0.250 mg/ml, less than 0.100 mg/ml, lessthan 0.050 mg/ml, or less than 0.010 mg/ml.

The pharmaceutically acceptable excipient can be an adjuvant in additionto the PD1 antibody or PDL1 antibody. The additional adjuvant can beother genes that are expressed in an alternative plasmid or aredelivered as proteins in combination with the plasmid above in thevaccine. The adjuvant may be selected from the group consisting of:α-interferon(IFN-α), β-interferon (IFN-β), β-interferon, plateletderived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growthfactor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelialthymus-expressed chemokine (TECK), mucosae-associated epithelialchemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 havingthe signal sequence deleted and optionally including the signal peptidefrom IgE. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, plateletderived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growthfactor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or acombination thereof.

Other genes that can be useful as adjuvants in addition to the PD1antibody or PDL1 antibody include those encoding: MCP-1, MIP-1a, MIP-1p,IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1,MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3,CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L,vascular growth factor, fibroblast growth factor, IL-7, IL-22, nervegrowth factor, vascular endothelial growth factor, Fas, TNF receptor,Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS,KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1,Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1,JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec,TRAILrecDRCS, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND,NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 andfunctional fragments thereof.

The vaccine may further comprise a genetic vaccine facilitator agent asdescribed in U.S. Serial No. 021,579 filed Apr. 1, 1994, which is fullyincorporated by reference. The vaccine can be formulated according tothe mode of administration to be used. An injectable vaccinepharmaceutical composition can be sterile, pyrogen free and particulatefree. An isotonic formulation or solution can be used. Additives forisotonicity can include sodium chloride, dextrose, mannitol, sorbitol,and lactose. The vaccine can comprise a vasoconstriction agent. Theisotonic solutions can include phosphate buffered saline. Vaccine canfurther comprise stabilizers including gelatin and albumin. Thestabilizers can allow the formulation to be stable at room or ambienttemperature for extended periods of time, including LGS or polycationsor polyanions.

3. Method of Vaccination

The present invention is also directed to a method of increasing animmune response in a subject. Increasing the immune response can be usedto treat and/or prevent disease in the subject. The method can includeadministering the herein disclosed vaccine to the subject. The subjectadministered the vaccine can have an increased or boosted immuneresponse as compared to a subject administered the antigen alone. Insome embodiments, the immune response can be increased by about 0.5-foldto about 15-fold, about 0.5-fold to about 10-fold, or about 0.5-fold toabout 8-fold. Alternatively, the immune response in the subjectadministered the vaccine can be increased by at least about 0.5-fold, atleast about 1.0-fold, at least about 1.5-fold, at least about 2.0-fold,at least about 2.5-fold, at least about 3.0-fold, at least about3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at leastabout 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, atleast about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold,at least about 8.0-fold, at least about 8.5-fold, at least about9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at leastabout 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, atleast about 12.0-fold, at least about 12.5-fold, at least about13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at leastabout 14.5-fold, or at least about 15.0-fold.

In still other alternative embodiments, the immune response in thesubject administered the vaccine can be increased about 50% to about1500%, about 50% to about 1000%, or about 50% to about 800%. In otherembodiments, the immune response in the subject administered the vaccinecan be increased by at least about 50%, at least about 100%, at leastabout 150%, at least about 200%, at least about 250%, at least about300%, at least about 350%, at least about 400%, at least about 450%, atleast about 500%, at least about 550%, at least about 600%, at leastabout 650%, at least about 700%, at least about 750%, at least about800%, at least about 850%, at least about 900%, at least about 950%, atleast about 1000%, at least about 1050%, at least about 1100%, at leastabout 1150%, at least about 1200%, at least about 1250%, at least about1300%, at least about 1350%, at least about 1450%, or at least about1500%.

The vaccine dose can be between 1 μg to 10 mg active component/kg bodyweight/time, and can be 20 μg to 10 mg component/kg body weight/time.The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, or 31 days. The number of vaccine doses for effective treatment canbe 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

a. Administration

The vaccine can be formulated in accordance with standard techniqueswell known to those skilled in the pharmaceutical art. Such compositionscan be administered in dosages and by techniques well known to thoseskilled in the medical arts taking into consideration such factors asthe age, sex, weight, and condition of the particular subject, and theroute of administration. The subject can be a mammal, such as a human, ahorse, a cow, a pig, a sheep, a cat, a dog, a rat, or a mouse.

The vaccine can be administered prophylactically or therapeutically. Inprophylactic administration, the vaccines can be administered in anamount sufficient to induce an immune response. In therapeuticapplications, the vaccines are administered to a subject in need thereofin an amount sufficient to elicit a therapeutic effect. An amountadequate to accomplish this is defined as “therapeutically effectivedose.” Amounts effective for this use will depend on, e.g., theparticular composition of the vaccine regimen administered, the mannerof administration, the stage and severity of the disease, the generalstate of health of the patient, and the judgment of the prescribingphysician.

The vaccine can be administered by methods well known in the art asdescribed in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997));Felgner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Felgner(U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S.Pat. No. 5,679,647, issued Oct. 21, 1997), the contents of all of whichare incorporated herein by reference in their entirety. The DNA of thevaccine can be complexed to particles or beads that can be administeredto an individual, for example, using a vaccine gun. One skilled in theart would know that the choice of a pharmaceutically acceptable carrier,including a physiologically acceptable compound, depends, for example,on the route of administration of the expression vector.

The vaccine can be delivered via a variety of routes. Typical deliveryroutes include parenteral administration, e.g., intradermal,intramuscular or subcutaneous delivery. Other routes include oraladministration, intranasal, and intravaginal routes. For the DNA of thevaccine in particular, the vaccine can be delivered to the interstitialspaces of tissues of an individual (Felgner et al., U.S. Pat. Nos.5,580,859 and 5,703,055, the contents of all of which are incorporatedherein by reference in their entirety). The vaccine can also beadministered to muscle, or can be administered via intradermal orsubcutaneous injections, or transdermally, such as by iontophoresis.Epidermal administration of the vaccine can also be employed. Epidermaladministration can involve mechanically or chemically irritating theoutermost layer of epidermis to stimulate an immune response to theirritant (Carson et al., U.S. Pat. No. 5,679,647, the contents of whichare incorporated herein by reference in its entirety).

The vaccine can also be formulated for administration via the nasalpassages. Formulations suitable for nasal administration, wherein thecarrier is a solid, can include a coarse powder having a particle size,for example, in the range of about 10 to about 500 microns which isadministered in the manner in which snuff is taken, i.e., by rapidinhalation through the nasal passage from a container of the powder heldclose up to the nose. The formulation can be a nasal spray, nasal drops,or by aerosol administration by nebulizer. The formulation can includeaqueous or oily solutions of the vaccine.

The vaccine can be a liquid preparation such as a suspension, syrup orelixir. The vaccine can also be a preparation for parenteral,subcutaneous, intradermal, intramuscular or intravenous administration(e.g., injectable administration), such as a sterile suspension oremulsion.

The vaccine can be incorporated into liposomes, microspheres or otherpolymer matrices (Felgner et al., U.S. Pat. No. 5,703,055; Gregoriadis,Liposome Technology, Vols. I to III (2nd ed. 1993), the contents ofwhich are incorporated herein by reference in their entirety). Liposomescan consist of phospholipids or other lipids, and can be nontoxic,physiologically acceptable and metabolizable carriers that arerelatively simple to make and administer.

The vaccine can be administered via electroporation, such as by a methoddescribed in U.S. Pat. No. 7,664,545, the contents of which areincorporated herein by reference. The electroporation can be by a methodand/or apparatus described in U.S. Pat. Nos. 6,302,874; 5,676,646;6,241,701; 6,233,482; 6,216,034; 6,208,893; 6,192,270; 6,181,964;6,150,148; 6,120,493; 6,096,020; 6,068,650; and 5,702,359, the contentsof which are incorporated herein by reference in their entirety. Theelectroporation may be carried out via a minimally invasive device.

The minimally invasive electroporation device (“MID”) may be anapparatus for injecting the vaccine described above and associated fluidinto body tissue. The device may comprise a hollow needle, DNA cassette,and fluid delivery means, wherein the device is adapted to actuate thefluid delivery means in use so as to concurrently (for example,automatically) inject DNA into body tissue during insertion of theneedle into the said body tissue. This has the advantage that theability to inject the DNA and associated fluid gradually while theneedle is being inserted leads to a more even distribution of the fluidthrough the body tissue. The pain experienced during injection may bereduced due to the distribution of the DNA being injected over a largerarea.

The MID may inject the vaccine into tissue without the use of a needle.The MID may inject the vaccine as a small stream or jet with such forcethat the vaccine pierces the surface of the tissue and enters theunderlying tissue and/or muscle. The force behind the small stream orjet may be provided by expansion of a compressed gas, such as carbondioxide through a micro-orifice within a fraction of a second. Examplesof minimally invasive electroporation devices, and methods of usingthem, are described in published U.S. Patent Application No.20080234655; U.S. Pat. No. 6,520,950; U.S. Pat. No. 7,171,264; U.S. Pat.No. 6,208,893; U.S. Pat. No. 6,009,347; U.S. Pat. No. 6,120,493; U.S.Pat. No. 7,245,963; U.S. Pat. No. 7,328,064; and U.S. Pat. No.6,763,264, the contents of each of which are herein incorporated byreference.

The MID may comprise an injector that creates a high-speed jet of liquidthat painlessly pierces the tissue. Such needle-free injectors arecommercially available. Examples of needle-free injectors that can beutilized herein include those described in U.S. Pat. Nos. 3,805,783;4,447,223; 5,505,697; and 4,342,310, the contents of each of which areherein incorporated by reference.

A desired vaccine in a form suitable for direct or indirectelectrotransport may be introduced (e.g., injected) using a needle-freeinjector into the tissue to be treated, usually by contacting the tissuesurface with the injector so as to actuate delivery of a jet of theagent, with sufficient force to cause penetration of the vaccine intothe tissue. For example, if the tissue to be treated is mucosa, skin ormuscle, the agent is projected towards the mucosal or skin surface withsufficient force to cause the agent to penetrate through the stratumcorneum and into dermal layers, or into underlying tissue and muscle,respectively.

Needle-free injectors are well suited to deliver vaccines to all typesof tissues, particularly to skin and mucosa. In some embodiments, aneedle-free injector may be used to propel a liquid that contains thevaccine to the surface and into the subject's skin or mucosa.Representative examples of the various types of tissues that can betreated using the invention methods include pancreas, larynx,nasopharynx, hypopharynx, oropharynx, lip, throat, lung, heart, kidney,muscle, breast, colon, prostate, thymus, testis, skin, mucosal tissue,ovary, blood vessels, or any combination thereof.

The MID may have needle electrodes that electroporate the tissue. Bypulsing between multiple pairs of electrodes in a multiple electrodearray, for example set up in rectangular or square patterns, providesimproved results over that of pulsing between a pair of electrodes.Disclosed, for example, in U.S. Pat. No. 5,702,359 entitled “NeedleElectrodes for Mediated Delivery of Drugs and Genes” is an array ofneedles wherein a plurality of pairs of needles may be pulsed during thetherapeutic treatment. In that application, which is incorporated hereinby reference as though fully set forth, needles were disposed in acircular array, but have connectors and switching apparatus enabling apulsing between opposing pairs of needle electrodes. A pair of needleelectrodes for delivering recombinant expression vectors to cells may beused. Such a device and system is described in U.S. Pat. No. 6,763,264,the contents of which are herein incorporated by reference.Alternatively, a single needle device may be used that allows injectionof the DNA and electroporation with a single needle resembling a normalinjection needle and applies pulses of lower voltage than thosedelivered by presently used devices, thus reducing the electricalsensation experienced by the patient.

The MID may comprise one or more electrode arrays. The arrays maycomprise two or more needles of the same diameter or differentdiameters. The needles may be evenly or unevenly spaced apart. Theneedles may be between 0.005 inches and 0.03 inches, between 0.01 inchesand 0.025 inches; or between 0.015 inches and 0.020 inches. The needlemay be 0.0175 inches in diameter. The needles may be 0.5 mm, 1.0 mm, 1.5mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more spaced apart.

The MID may consist of a pulse generator and a two or more-needlevaccine injectors that deliver the vaccine and electroporation pulses ina single step. The pulse generator may allow for flexible programming ofpulse and injection parameters via a flash card operated personalcomputer, as well as comprehensive recording and storage ofelectroporation and patient data. The pulse generator may deliver avariety of volt pulses during short periods of time. For example, thepulse generator may deliver three 15 volt pulses of 100 ms in duration.An example of such a MID is the Elgen 1000 system by Inovio BiomedicalCorporation, which is described in U.S. Pat. No. 7,328,064, the contentsof which are herein incorporated by reference.

The MID may be a CELLECTRA (Inovio Pharmaceuticals, Plymouth Meeting,Pa.) device and system, which is a modular electrode system, thatfacilitates the introduction of a macromolecule, such as a DNA, intocells of a selected tissue in a body or plant. The modular electrodesystem may comprise a plurality of needle electrodes; a hypodermicneedle; an electrical connector that provides a conductive link from aprogrammable constant-current pulse controller to the plurality ofneedle electrodes; and a power source. An operator can grasp theplurality of needle electrodes that are mounted on a support structureand firmly insert them into the selected tissue in a body or plant. Themacromolecules are then delivered via the hypodermic needle into theselected tissue. The programmable constant-current pulse controller isactivated and constant-current electrical pulse is applied to theplurality of needle electrodes. The applied constant-current electricalpulse facilitates the introduction of the macromolecule into the cellbetween the plurality of electrodes. Cell death due to overheating ofcells is minimized by limiting the power dissipation in the tissue byvirtue of constant-current pulses. The Cellectra device and system isdescribed in U.S. Pat. No. 7,245,963, the contents of which are hereinincorporated by reference.

The MID may be an Elgen 1000 system (Inovio Pharmaceuticals). The Elgen1000 system may comprise device that provides a hollow needle; and fluiddelivery means, wherein the apparatus is adapted to actuate the fluiddelivery means in use so as to concurrently (for example automatically)inject fluid, the described vaccine herein, into body tissue duringinsertion of the needle into the said body tissue. The advantage is theability to inject the fluid gradually while the needle is being insertedleads to a more even distribution of the fluid through the body tissue.It is also believed that the pain experienced during injection isreduced due to the distribution of the volume of fluid being injectedover a larger area.

In addition, the automatic injection of fluid facilitates automaticmonitoring and registration of an actual dose of fluid injected. Thisdata can be stored by a control unit for documentation purposes ifdesired.

It will be appreciated that the rate of injection could be either linearor non-linear and that the injection may be carried out after theneedles have been inserted through the skin of the subject to be treatedand while they are inserted further into the body tissue.

Suitable tissues into which fluid may be injected by the apparatus ofthe present invention include tumor tissue, skin or liver tissue but maybe muscle tissue.

The apparatus further comprises needle insertion means for guidinginsertion of the needle into the body tissue. The rate of fluidinjection is controlled by the rate of needle insertion. This has theadvantage that both the needle insertion and injection of fluid can becontrolled such that the rate of insertion can be matched to the rate ofinjection as desired. It also makes the apparatus easier for a user tooperate. If desired means for automatically inserting the needle intobody tissue could be provided.

A user could choose when to commence injection of fluid. Ideallyhowever, injection is commenced when the tip of the needle has reachedmuscle tissue and the apparatus may include means for sensing when theneedle has been inserted to a sufficient depth for injection of thefluid to commence. This means that injection of fluid can be prompted tocommence automatically when the needle has reached a desired depth(which will normally be the depth at which muscle tissue begins). Thedepth at which muscle tissue begins could for example be taken to be apreset needle insertion depth such as a value of 4 mm which would bedeemed sufficient for the needle to get through the skin layer.

The sensing means may comprise an ultrasound probe. The sensing meansmay comprise a means for sensing a change in impedance or resistance. Inthis case, the means may not as such record the depth of the needle inthe body tissue but will rather be adapted to sense a change inimpedance or resistance as the needle moves from a different type ofbody tissue into muscle. Either of these alternatives provides arelatively accurate and simple to operate means of sensing thatinjection may commence. The depth of insertion of the needle can furtherbe recorded if desired and could be used to control injection of fluidsuch that the volume of fluid to be injected is determined as the depthof needle insertion is being recorded.

The apparatus may further comprise: a base for supporting the needle;and a housing for receiving the base therein, wherein the base ismoveable relative to the housing such that the needle is retractedwithin the housing when the base is in a first rearward positionrelative to the housing and the needle extends out of the housing whenthe base is in a second forward position within the housing. This isadvantageous for a user as the housing can be lined up on the skin of apatient, and the needles can then be inserted into the patient's skin bymoving the housing relative to the base.

As stated above, it is desirable to achieve a controlled rate of fluidinjection such that the fluid is evenly distributed over the length ofthe needle as it is inserted into the skin. The fluid delivery means maycomprise piston driving means adapted to inject fluid at a controlledrate. The piston driving means could for example be activated by a servomotor. However, the piston driving means may be actuated by the basebeing moved in the axial direction relative to the housing. It will beappreciated that alternative means for fluid delivery could be provided.Thus, for example, a closed container which can be squeezed for fluiddelivery at a controlled or non-controlled rate could be provided in theplace of a syringe and piston system.

The apparatus described above could be used for any type of injection.It is however envisaged to be particularly useful in the field ofelectroporation and so it may further comprises means for applying avoltage to the needle. This allows the needle to be used not only forinjection but also as an electrode during, electroporation. This isparticularly advantageous as it means that the electric field is appliedto the same area as the injected fluid. There has traditionally been aproblem with electroporation in that it is very difficult to accuratelyalign an electrode with previously injected fluid and so user's havetended to inject a larger volume of fluid than is required over a largerarea and to apply an electric field over a higher area to attempt toguarantee an overlap between the injected substance and the electricfield. Using the present invention, both the volume of fluid injectedand the size of electric field applied may be reduced while achieving agood fit between the electric field and the fluid.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

EXAMPLES Example 1

Mice were immunized two times at two week intervals as three separategroups: vector pVAX only, DNA vaccine (HPV) only, and DNA vaccine (HPV)combined with mAb PDL1. For the combination, a mAb PD1 L was deliveredbeginning on day 10 post-first immuniziation, and thereafter every threedays until mice were sacrificed 8 days after last immunization. Thedata, as shown in the bar graph in FIG. 1, shows a 30% increase in Tcell responses induced by co-therapy with anti PDL1 antibody.

The PDL1 mAb can be generated or can be obtained commercially, e.g.,CD274 (B7-H1, PD-L1) Rat Anti-Mouse mAb (clone 10F.9G2), PE-Cy®7conjugate (Life Technologies).

Example 2

mAb

The PDL1, PD1, TIM-3 and LAG-3 mAb can be generated or can be obtainedcommercially, e.g., CD274 (B7-H1, PD-L1) Rat Anti-Mouse mAb (clone10F.9G2), PE-Cy®7 conjugate (Life Technologies or Bio X cell), ratanti-mouse PD-1 (clone RMP1-14 or J43), rat anti-mouse TIM-3 (CloneRMT3-23; Bio X Cell), rat anti-mouse LAG-3 (Clone C9B7W; BIO X Cell),respectively.

Mouse Immunization

C57BL/6 mice (n=4) were immunized thrice, with a two-week intervalbetween immunizations, with 25 μg hTERT construct with or without thedelivery of either PD1, TIM3 and LAG3 mAb's.

Immunization Groups

Group II-hTERT

Group III-hTERT+PD1

Group IV-hTERT+TIM3

Group V-hTERT+LAG3

Each one of the mAb was delivered 3 days post-second immunization withhTERT.

The data represented in the graphs in FIG. 2-4 show that the blockade ofimmune checkpoints after boost increases immune responses for anti PD-1,anti-TIM3, and anti-LAG3 antibodies. Induction of enhanced IFNγproduction of hTERT-specific CD8+ T cells following DNA immunizationwith immune checkpoint inhibitors: Cytokine-recall responses to hTERTantigen were measured one week after last immunization by ICS and flowcytometry. The right plot graphs depict the total hTERT-specific CD8+ Tcells expressing total IFNγ for mice treated with PD1 (FIG. 2), TIM3(FIG. 3), and LAG3 (FIG. 4). The left plot graphs show the percentagesof hTERT-specific CD3+CD8+ T cells displaying double release of thecytokines IFNγ and TNFα (PD1 in FIG. 2, TIM3 in FIG. 3, and LAG3 in FIG.4. Experiments were performed independently at least twice and datarepresent the mean±SEM of four mice per group.

The results show surprisingly that the anti-TIM3 and anti-LAG3antibodies produced significantly higher increase in immune response inthe subjects.

Example 3

Timing of Checkpoint Inhibitor Delivery relative to Vaccination

Delivery of checkpoint inhibitors after prime immunization.

(A)Early Delivery:

-   -   C57BL/6 mice (n=3-4) were immunized twice, with a two-week        interval between immunizations, with 25 μg hTERT construct with        or without the delivery of either PD1, TIM3 and LAG3 mAb's. The        initial mAb delivery of checkpoint inhibitors was at day 10        (D10), then D13, D16, and D19.

(B) Late Delivery

-   -   C57BL/6 mice (n=4) were immunized three times at three week        intervals with 25 μg hTERT construct with or without the        delivery of either PD1, TIM3, and LAG3 mAb's. The initial mAb        delivery was at 3 days post-second immunization, and        administered thereafter every three days until sacrificed.

Delivery of blockade immune checkpoint inhibitors is time sensitive.IFNγ-specific recall responses to hTERT antigen were measured one weekafter final immunization by flow cytometry during early delivery of mAbversus later delivery of mAb checkpoints. The plot graphs in FIG. 5represents results for depicts the hTERT-specific CD8 T cells expressingtotal IFNγ for mice treated with or without PD1, TIM3 and LAG3 soonafter priming immunization. The plot graphs in FIG. 6 depicts thehTERT-specific CD8 T cells expressing total IFNγ for mice treated withor without PD1, TIM3 and LAG3 soon after boost immunization. Thus, thedata shows that late delivery yields surprising results in drivingantigen specific T cell expansion by checkpoint inhibitors when comparedwith early delivery, which shows slower antigen specific immunity.

1. A composition for enhancing an immune response against an antigen ina subject in need thereof, comprising: a) TIM-3 antibody or LAG-3antibody, and b) a synthetic antigen capable of generating an immuneresponse in the subject, or a biologically functional fragment orvariant thereof.
 2. The composition of claim 1 wherein the syntheticantigen is an isolated DNA that encodes for the antigen.
 3. Thecomposition of claim 2 wherein the synthetic antigen is selected fromthe group consisting of: hTERT, prostate, WT1, tyrosinase, NYES01,PRAME, MAGE, CMV, herpes, HIV, HPV, HCV, HBV, influenza, RSV, Plasmodiumfalciparum, and C. difficle.
 4. The composition of claim 3, wherein theHPV antigen is E6 and E7 domains of subtypes selected from the groupconsisting of: HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV52, andHPV58, and a combination thereof.
 5. The composition of claim 3, whereinthe HIV antigen is selected from the group consisting of: Env A, Env B,Env C, Env D, B Nef-Rev, and Gag, and a combination thereof.
 6. Thecomposition of claim 3, wherein the influenza antigen is selected fromthe group consisting of: H1 HA, H2 HA, H3 HA, H5 HA, BHA antigen, andany combination thereof.
 7. The composition of claim 3, wherein thePlasmodium falciparum antigen includes a circumsporozoite (CS) antigen.8. The composition of claim 3, wherein the C. difficle antigen isselected from the group consisting of: Toxin A, and Toxin B, and acombination thereof.
 9. The composition of claim 3, wherein the HCVantigen is selected from the group consisting of: E1, E2, NS3, NS4a,NS4b, NS5a, and NS5b, and a combination thereof.
 10. The composition ofclaim 3, wherein the HBV antigen is selected from the group consistingof: surface antigen type A, surface antigen type B, surface antigen typeC, surface antigen type D, surface antigen type E, surface antigen typeF, surface antigen type G, surface antigen type H, and core antigen, anda combination thereof.
 11. The composition of claim 3, wherein the RSVantigen is selected from the group consisting of: F, G, NS1, NS2, N, M,M2-1, M2-2, P, SH, and L protein, and a combination thereof.
 12. Thecomposition of claim 3, wherein the synthetic antigen is hTERT.
 13. Thecomposition of claim 3, wherein the prostate antigen is selected fromthe group consisting of: PSA, PSMA, STEAP, PSCA, and PAP, and acombination thereof.
 14. The composition of claim 3, wherein thesynthetic antigen is WT1 antigen.
 15. The composition of claim 3,wherein the synthetic antigen is tyrosinase.
 16. The composition ofclaim 3, wherein the synthetic antigen is NYES01.
 17. The composition ofclaim 3, wherein the synthetic antigen is PRAME.
 18. The composition ofclaim 1, further comprising a pharmaceutically acceptable excipient. 19.A method for increasing an immune response in a subject in need thereof,the method comprising administering the composition of claim 1 to thesubject.
 20. The method of claim 19, wherein administering thecomposition comprises an electroporating step.
 21. A method ofincreasing an immune response in a subject in need thereof byadministering a combination of synthetic antigen and checkpointinhibitor, wherein the administering step comprises: administering tothe subject a prime vaccination and a boost vaccination of syntheticantigen, and subsequent to the boost vaccination, administering to thesubject a checkpoint inhibitor.
 22. The method of claim 21, furthercomprising a step of further administering to the subject a subsequentboost vaccination of the synthetic antigen.
 23. The method of claim 22,wherein any of the administering steps include deliveringelectroporation to site of administration.